VACCINE

Abstract: A non-naturally occurring variant of a C-terminal fragment of a Plasmodium merozoite surface protein-1 (MSP-1) wherein said variant has (i) a reduced affinity, compared with a naturally occurring Plasmodium MSP-119, for at least one first antibody capable of blocking the binding of a second antibody, which second antibody inhibits the proteolytic cleavage of Plasmodium MSP-142 and (ii) substantially the same affinity for at least one third antibody compared with said naturally occurring Plasmodium MSP-119, which third antibody inhibits the proteolytic cleavage of <i>Plasmodium MSP-142 is provided for use in an anti-malarial vaccine.

Description: VACCINE Field of the Invention The present invention relates to modified Plasmodium MSP-1 protein variants and their use in producing a vaccine against malaria. It also relates to a method for the rational design of suitable variants. Background to the Invention Malaria is a devastating disease that causes widespread morbidity and mortality in areas where it is transmitted by anopheline mosquitoes. In areas of high transmission young children and non-immune visitors are most at risk from this disease, which is caused by protozoa of the genus Plasmodium. In areas of lower or unstable transmission, epidemics of the disease can result and afflict individuals of all ages. The most dangerous form of malaria, responsible for much of the morbidity and most of the mortality, is caused by the species Plasmodium falciparum. It has been estimated that 2 billion people are at risk from malaria, with 200-300 million clinical cases and 1-2 million deaths each year. The parasite has a complex life cycle in its human and mosquito hosts. In humans the stage of the life cycle which is responsible for the clinical symptoms of the disease occurs in the bloodstream. During this phase the parasite is largely hidden within host red blood cells. Here the parasite grows and multiplies. For example, within a red blood cell each P. falciparum parasite divides several times to produce approximately 20 new ones during a 48 hour cycle. At this point the red blood cell is burst open and the parasites (called merozoites at this stage) are released into the bloodstream. The merozoites must enter new red blood cells in order to survive and for the cycle of replication in the blood to continue. If the parasites do not manage to enter red blood cells they cannot survive for very long and are rapidly destroyed. Symptoms of malaria such as fever are associated with this cvclic merozoite release and re-invasion of red blood cells. There is an urgent need for a vaccine against malaria. There is no effective vaccine currently available. In addition, mosquito control " >y the spraying of residual insecticides is either becoming ineffective or considered to be unacceptable, and there is a very worrying spread of drug resistance within parasites. The rapid spread of drug resistance is worrying because compounds such as the cheap and once-effective chloroquine are no longer useful in many parts of the world, and there are few if any new drugs available that are both cheap and effective. Vaccines against microorganisms can be very cost effective and efficient ways to protect populations against infectious diseases. Because of the complexity of the parasite's life cycle there are a number of points in its development within humans that could be the target of a protective immune response. It is known that with increasing age and exposure individuals do become immune to malaria, suggesting that protective responses do develop with time. Broadly speaking there are three types of vaccine strategy: to target the pre-erythrocytic stages, the asexual blood stage and the sexual stage. The pre-erythrocytic stages are the sporozoites that are injected by an infected mosquito when it takes a blood meal and the initial development of the parasite in the liver. The asexual blood stage is the infection and release of merozoites from red blood cells that occurs in a cyclic manner, and the stage responsible for the manifestation of the clinical symptoms. The sexual stage takes place in the mosquito's gut after it has ingested gametocytes in a blood meal and this initiates the infection of the insect to complete the cycle; a vaccine against the sexual stages would not protect the individual but could reduce transmission and therefore the incidence of malaria in a given human population. During the asexual cycle in the blood the parasite is directly exposed to the host's immune system, and in particular to antibodies circulating within the bloodstream, only transiently: when merozoites are released by rupture of one cell and before they penetrate another. If there are specific antibodies that can bind to the surface of the parasite then it is possible that these antibodies will interfere with the ability of the parasite to invade a new red blood cell. In fact it has been shown that several monoclonal antibodies that recognise single epitopes on parasite surface proteins, are capable of neutralising the parasite and preventing the cycle of reproduction within red blood cells. One of the best characterised proteins on the surface of the merozoite is called merozoite surface protein 1 (MSP-1). MSP-1 is a large protein that varies in size and amino acid sequence in different parasite lines. It is synthesised as a precursor molecule of -200 kDa by the intracellular parasite and located on the parasite's surface. During release of merozoites from red blood cells and the re-invasion of new erythrocytes the protein undergoes at least two proteolytic modifications. In the first modification as a result of a process called primary processing, the precursor is cleaved to four fragments of ~83, 30, 38 and 42 kDa that remain together as a complex on the merozoite surface. This complex also contains two other proteins of 22 kDa and 36 kDa derived from different genes. The complex is maintained by non-covalent interactions between the different subunits and is held on the merozoite surface by a glycosyl phosphatidyl inositol anchor, attached to the C -terminus of the 42 kDa fragment and inserted into the plasma membrane of the merozoite. At the time of merozoite invasion of an erythrocyte the C-terminal 42 kDa fragment is cleaved by a second proteolytic cleavage in a process called secondary processing. The result of secondary processing is that the entire complex is shed from the surface of the merozoite except for a C-terminal sub-fragment that consists of just under one hundred amino acids and which is carried into the newly invaded erythrocyte on the surface of the merozoite. Based on sequence similarities, the structure of this small C-terminal fragment (called MSP-1 19 ) was suggested to consist of two epidermal growth factor (EGF)-like domains (see sequence in Figure 1) (Blackman et al., 1991). An EGF-like motif consists of a 45-50 amino acid sequence with a characteristic disulphide bonding pattern and such domains occur frequently in extracellular modular proteins of animals. In the MSP-1 C-terminal fragment each of the motifs contains six Cys residues proposed to form three disulphide bonds and each motif has a partial match to the EGF consensus (see Figure 1). However, because the degree of similarity is limited and since the pattern of its disulphide bonding is not known, the designation of the MSP-1 C-terminal fragment as comprised of EGF- like structures has been regarded as tentative. Other relatively divergent potential EGF- like sequences occur in Plasmodium proteins, but previous structure determinations have been confined to those from metazoan organisms (Campbell et al, 1998). A number of studies have implicated MSP-1 as the target of a protective immune response. Although the goal of this work is to develop a malaria vaccine for use in humans, out of necessity most of this experimental work has been done either in model animal systems or in vitro. These include studies of the effect of specific antibodies on parasite invasion of erythrocytes in vitro, passive immunisation studies in rodent malaria models in laboratory mice and direct immunisation in both rodent and primate malaria models using either native protein (derived from the parasite) or recombinant protein expressed from parts of the MSP-1 gene in heterologous organisms. Sero-epidemiological studies have also showed a correlation between human antibody responses to parts of the MSP-1 molecule and protection against clinical disease. Much, but not all, of the work has focused on the immune response to the C-terminal MSP-1 1 . For example some monoclonal antibodies that recognise MSP-1 19 prevent red blood cell invasion in in vitro cultures (Blackman et al.. 1990). Interestingly, these antibodies that inhibit invasion also inhibit the secondary processing of the 42 kDa fragment, suggesting the mechanism by which they work is by steric hinderance of the protease responsible for secondary processing (Blackman et al, 1994). Since secondary processing goes to completion during successful invasion, if it cannot occur then invasion is interrupted. All of the work described above would suggest ' that MSP-1 and in particular polypeptides based on the C-terminal sequence that forms the 42 kDa or the MSP-1 19 region, should be very good candidates for malaria vaccine development. However, several studies have shown that the epitopes or binding sites for antibodies on MSP-1 19 require a correct polypeptide tertiary structure, and that this is destroyed by treatments that reduce the disulphide bonds that are postulated to be present between the cysteine residues present in MSP-1 19 . This limitation appears to have been overcome by the expression of recombinant protein in ways that allow antibodies that recognise the native parasite MSP- 1 to bind. Other investigators have suggested that other parts of MSP-1 also have potential for inclusion in a vaccine, however the MSP-1 C-terminal fragment is currently the lead candidate for development of a vaccine against the blood stages of the malaria parasite (Diggs et al.. 1993; Stoute et α/.,L998). As stated above, every -48 hours P. falciparum merozoites are released from the infected erythrocyte to re-invade new red blood cells and during this time they are exposed to the host ' s immune system. Therefore, the question arises as to how the parasite has evolved to avoid the potentially lethal effects of, for example, neutralising antibodies. In other infectious micro-organisms it is clear that there is a constant battle between the immune system and the micro-organism, and that sophisticated mechanisms have been evolved by micro-organisms to evade the immune response. For example antigenic variation and antigenic diversity are two mechanisms that involve presenting the immune system with "a moving target" such that even though an immune response to one variant of the micro- organism may kill that variant, new variants are produced that are at least partially or fully resistant to the immune response. In the case of malaria merozoites and in particular MSP-1 an alternative mechanism has been proposed whereby the binding of some antibodies ("blocking antibodies") can prevent the binding of neutralising antibodies and thereby allow the parasite to successfully invade a red blood cell even in the presence of neutralising antibodies (Guevara Patiho et al., 1997). These blocking antibodies may be of two types, those against epitopes that are formed from amino acids that are distant in the linear primary sequence from the epitopes that are the target of neutralising antibodies, and those that are against epitopes that overlap with the epitopes of the neutralising antibodies. This represents a novel mechanism by which a parasite can evade an effective immune response, and unlike mechanisms based on antigenic polymorphism or diversity, it is not dependent upon amino acid sequence diversity. Some monoclonal antibodies (mAbs) that bind to MSP-11 9 inhibit the proteolytic cleavage and erythrocyte invasion, suggesting that cleavage is a prerequisite for invasion (Blackman et al., 1994). Other mAbs that bind to the MSP-1 C-terminal fragment do not inhibit processing or invasion but block the binding of the inhibitory neutralizing antibodies. Other antibodies that bind to MSP-119 neither inhibit nor block the binding of inhibitory antibodies. In the presence of blocking antibodies, inhibitory antibodies are ineffective and invasion proceeds. The balance between inhibitory and blocking antibodies induced by immunisation may be a critical factor in determining whether or not the immune response is effective in preventing invasion (Guevara Patiho et al. 1997). Summarv of the Invention An object of the present invention is therefore to provide an effective vaccine against the malaria parasite based on variants of the Plasmodium MSP-1 protein. In designing such a vaccine, the following criteria should be met: 1. The amino acid sequence of the polypeptide to be used in the vaccine should contain epitopes that are the targets of, and can induce, neutralising antibodies. 2. The polypeptide should ideally not include amino acid sequences that only form epitopes for blocking antibodies. 3. If the polypeptide contains epitopes for both neutralising and blocking antibodies then it should be modified to remove the blocking antibody epitopes without affecting the neutralising epitopes. To assist in the design of candidate vaccine polypeptides fulfilling these three criteria, it is important to determine the three-dimensional structure of the MSP-1 C-terminal fragment since this will help in mapping sites of antibody interactions with this fragment. We have therefore determined the solution structure of the MSP-1 C-terminal. including the pattern of disulphide bonding, using NMR techniques. We have made amino acid substitutions in the sequence of MSP-1 19 that prevent the binding of individual blocking monoclonal antibodies, without affecting the binding of neutralising antibodies. By determining the 3-dimensional structure of MSP-1 19 we have identified where these antibody binding sites are located in the tertiary structure and this has allowed other amino acid substitutions to be made that have similar properties. We have shown that several substitutions, each affecting the binding of one or more blocking antibodies can be combined into a single molecule, and that these modified molecules continue to bind the neutralising antibodies but fail to bind any of the blocking antibodies. Such modified molecules are expected tαbe much more effective than the natural or wild- type protein structure at inducing a protective neutralising antibody response when used to immunise individuals as a malaria vaccine. In addition we have made other modifications in the primary structure of the molecule which do " not affect the binding of the neutralising antibodies but which may contribute to increased immunogenicity of the molecule. The modified MSP-1 1 structures, either alone or coupled to other carriers, which may or may not contain other parts of MSP-1 to enhance the immunogenicity (for example a combination of the remainder of the MSP-1 kDa fragment with the modified MSP-1 19 ) and provide additional T cell epitopes, would be more effective vaccines than equivalent structures that have not been modified in this way. Accordingly, the present invention provides a non-naturally occurring variant of a C-terminal fragment of a Plasmodium merozoite surface protein- 1 (MSP-1) wherein said variant has (i) a reduced affinity, compared with a naturally occurring Plasmodium MSP-1 1 9, for at least one first antibody capable of blocking the binding of a second antibody, which second antibody inhibits the proteolytic cleavage of Plasmodium MSP-1 2 and (ii) substantially the same affinity for said second antibody compared with said naturally occurring Plasmodium MSP-1 19 . Preferably, the Plasmodium MSP-1 1 and MSP-1 2 are Plasmodium falciparum MSP-1 19 The first antibody is preferably selected from mAbs IE1, 2.2, 7.5, 9C8 and 1 11.4. The second antibody is preferably selected from mAbs 12.8, 12.10 and 5B1. The present invention further provides a non-naturally occurring variant of a C-terminal fragment of a Plasmodium merozoite surface protein- 1 (MSP-1) comprising an amino acid modification at any one of amino acid residues 14, 15, 27, 31, 34 , 43 48 and 53 of the Plasmodium falciparum MSP-l ι amino acid sequence shown as SEQ I.D. No. 1 or their equivalent positions in other Plasmodium MSP-1 1 polypeptides. Preferably said modifications are substitutions selected from GlnM→Arg, Glnl4- Gly, Asnl 5→Arg, Glu27→Tyr, LeuS l→Arg, Tyr34→Ser, Tyr34→Ile, Glu43→Leu, Thr48— Lys and Asn53→Arg and their equivalents in other Plasmodium MSP-1 1 polypeptides. More preferably said substitutions are combinations of substitutions selected from [Glu27— Tyr. Leu31— »Arg and Glu43→Leu], [Glu27→Tyr. Leu31→Arg. Tyr34→Ser and Glu43→Leu], [Asnl5→Arg, Glu27→Tyr, Leu31→Arg and Glu43—»Leu] and their equivalents in other Plasmodium MSP-1 1 polypeptides. In a preferred embodiment, a variant MSP-1 polypeptide of the invention further comprises a mutation at Cysl2 and/or Cys28 of the Plasmodium falciparum MSP-1 19 amino acid sequence shown as SEQ I.D. No. 1. Preferably such modifications are substitutions selected from Cysl2-→Ile and Cys28— » rp, and Cysl2-→Ala and Cys28-→Phe. Most preferably the substitutions are combinations selected from [Cysl2— »Ile, Asn 15→Arg, Glu27-→Tyr, Cys28→Trp, Leu31-→Arg, Glu43-→Leu], [Cysl2→Ile, Asn 15-→Arg, Glu27→Tyr, Cys28→Trp, Leu31→Arg, Glu43→Leu, Asn53→Arg], [Cysl2→Ile, Asn 15→Arg, Glu27→Tyr, Cys28→Trp, Leu31→Arg, Tyr34→Ser, Glu43— >Leu. Asn53— ÷Arg] and their equivalents in other PlasmodiumMST > -\ \ς, polypeptides. The present invention also provides a method for producing a Plasmodium MSP-1 variant for use in preparing a vaccine composition which method comprises modifying one or more amino acid residues of a Plasmodium MSP-1 C-terminal fragment such that the resulting derivative has (i) a reduced affinity, compared with a naturally occurring Plasmodium MSP-1 19 , for at least one first antibody capable of blocking the binding of a second antibody, which second antibody inhibits the proteolytic cleavage of Plasmodium MSP-1 42 and (ii) substantially the same affinity for said second antibody compared with said naturally occurring Plasmodium MSP-1 19 . In particular the method of the invention preferably comprises as a preliminary step, selecting a candidate amino acid residue by reference to a three dimensional NMR model structure, preferably as set out in Table 2. More specifically, the 3D model structure is used to select a surface exposed amino acid residue. Advantageously, a further step is included of computer modelling the three dimensional structure of the variant to exclude polypeptides that do not fold correctly. The present invention also provides a non-naturally occurring Plasmodium MSP-1 variant obtained by the method of the invention. In a further aspect, the present invention provides a polynucleotide encoding a variant of the invention operably linked to a regulatory sequence capable of directing the expression of said nucleotide in a host cell. The polynucleotide may comprise a sequence which has been optimised for expression in the host cell. The host cell may be a Pichia pastoris cell. Also provided is a nucleic acid vector comprising a polynucleotide of the invention, including viral vectors, and a host cell comprising a nucleotide or vector of the invention. In another aspect, the present invention provides a pharmaceutical composition comprising a variant of the invention, a polynucleotide of the invention or a vector of the invention together with a pharmaceutically acceptable carrier or diluent. Preferably, the composition further comprises an immunogenic Plasmodium polypeptide or fragment or derivative thereof such as MSP-1 3 or a fragment or derivative thereof which may be covalently attached to the non-naturally occuring MSP-1 19 . It is preferred not to use wild-type MSP-1 19 sequences. The further immunogenic peptide may itself be derivatised in an analogous manner as described above for MSP-1 19 . Thus, epitopes present in the peptide may be identified and modified to prevent binding of blocking antibodies, without affecting the binding of neutralising antibodies. These epitopes may be capable of binding to antibodies which have similar properties to the first antibody described above, for example, in binding affinity. The further immunogenic peptide may comprise several such modifications in its amino acid sequence. The present invention also provides a method for producing anti-MSP-1 antibodies which method comprises administering a polypeptide variant of the invention, or a polynucleotide of the invention or a vector of the invention to a mammal, typically a non- human mammal. In a preferred embodiment, the present invention provides a method for producing polyclonal anti-MSP-1 antibodies which method comprises administering a polypeptide variant of the invention, or a polynucleotide of the invention or a vector of the invention to a mammal, typically a non-human mammal, and extracting the serum from said mammal. Also provided is an antibody produced by the said methods. The polypeptides. nucleotides and vectors of the present invention may be used in methods of treating and/or preventing malaria caused by Plasmodium species, in particular Plasmodium falciparum. Accordingly, the present invention provides a method of inducing immunity against malaria induced by Plasmodium falciparum which comprises administering to a person in need of such immunity an effective amount of a variant, a polynucleotide or a vector of the invention. Also provided is a method of immunizing a mammal, said method comprising administering an effective amount of a variant, a polynucleotide or a vector of the invention. In particular, said mammal is immunized against malaria. Preferably the mammal is a human. The present invention also provides a method of treating a malaria infection in a human patient which comprises administering to the patient an effective amount of the pharmaceutical composition of the invention. We further provide according to the present invention a nucleic acid encoding a Plasmodium MSP-1 polypeptide, in which the nucleic acid is optimised for expression in a heterologous host cell.. Preferably, the heterologous host is a Pischia pastoris cell. The MSP-1 polypeptide may be selected from the group comprising an MSP-1 42 polypeptide comprising a sequence shown in Figures 2C and 2E. an MSP-1 !9 polypeptide comprising a sequence shown in Figure 2C, and an MSP-1 3 polypeptide comprising a sequence shown in Figure 2E. The optimised nucleic acid may comprise a sequence selected from the sequences of Figure 2A. Figure 2B and Figure 2D. We further provide a vector comprising such a nucleic acid, a host cell comprising such a vector, and a pharmaceutical composition comprising such a nucleic acid or a vector, together with a pharmaceutically acceptable carrier or diluent. The pharmaceutical composition may further comprise an immunogenic Plasmodium polypeptide or fragment or derivative thereof. Detailed description of the invention Although in general the techniques mentioned herein are well known in the art. reference may be made in particular to Sambrook et al. Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al, Current Protocols in Molecular Biology (1995), John Wiley & Sons. Inc. A. MSP- 1 variant polypeptides The variant MSP-1 polypeptides of the present invention will be described with reference to Plasmodium falciparum MSP-1 amino acid sequences. However, it should be appreciated that except where otherwise stated, all references to MSP-1 polypeptides include homologues of MSP-1 found in other Plasmodium species, such as P. vivax, P. malariae and P. ovale which all infect humans and P. yoelii which infects mice. The variant MSP-1 polypeptides of the present invention are based on C-terminal fragments of the Plasmodium falciparum MSP-1 42 polypeptide shown as SEQ I.D. Nos. 2 or 3. Such polypeptides will comprise some or all of the MSP-11 region (SEQ I.D. No. 1), preferably at least substantially all of the domain 1 and/or domain 2 EGF-like sequences found in MSP-1 19 (approximately amino acids 1-47 and amino acids 48-96, respectively, of SEQ I.D. No. 1). It is particularly preferred to use regions that are conserved in most, more preferably all parasites of a single species to increase the effectiveness of the variant as a vaccine against a wide range of strains. Variant MSP-1 polypeptides of the present invention comprise modifications to their primary amino acid sequence that reduce the ability of blocking antibodies to bind to the MSP-1 polypeptides. In addition, any modifications made should maintain epitopes recognised by neutralising antibodies such that the affinity of the neutralising antibodies for the MSP-1 variant is substantially the same as for naturally-occurring MSP-1 polypeptides (such as an MSP-1 42 polypeptide having the sequence shown in SEQ I.D. Nos. 2 or 3). Some reduction in the binding of some neutralising antibodies may be tolerated since the primary objective is to inhibit the binding of blocking antibodies and it is likely that an effective reduction in the binding 'of blocking antibodies will compensate in terms of overall vaccine efficacy for a small reduction in neutralising antibody binding. 5 Neutralising antibodies in the context of the present invention are antibodies that inhibit malaria parasite replication. A variety of neutralising antibodies, polyclonal and monoclonal, are known in the art, including mAbs 12.8. 12.10 and 5B1 referred to in the Examples. The activity of neutralising antibodies can be determined in a variety of ways that have been described in the art. For example, a convenient assay method described in 10 Blackman et al, 1994 involves using preparations of merozoites (Blackman et al, 1993; Mrema et al. 1982) to measure cleavage of MSP-1 42 into MSP-1 33 and MSP-1 [9 . Briefly, freshly isolated merozoites are washed in ice-cold buffer and divided up into aliquots of about 2xl0 9 merozoites. A test antibody is added to each aliquot and the sample incubated at 37°C for 1 hour. The samples are then subjected to SDS-PAGE under non- 15 reducing conditions on a 12.5% polyacrylamide gel, Western blotted and the blot probed with antiserum to MSP-1 33 . In the control sample, two main bands are seen - one corresponding to MSP-1 42 and one lower molecular weight band corresponding to MSP-1 33 . Neutralising antibodies will reduce the amount of the lower molecular weight band as a result of inhibiting secondary proteolytic processing of MSP-1 42 . 20 This method is a particularly preferred method for assessing the efficacy of neutralising antibodies in the presence of antibodies believed to act as blocking antibodies. Where candidate competing blocking antibodies are to be tested, the merozoite sample is preincubated with a blocking antibody for 15 mins on ice prior to incubation _with a 25 neutralising antibody at 37°C for 1 hour as described above. Thus blocking antibodies can readily be identified and/or characterised using such an assay method. Other assay methods include merozoite invasion inhibition tests as described in Blackman et al, 1990. J 0 As discussed above, blocking antibodies are defined in the context of the present invention as antibodies that inhibit the binding of neutralising antibodies to MSP-1 but which do not themselves inhibit invasion of red blood cells by malaria parasites. Thus they "block" the neutralising function of the neutralising antibodies. A variety of blocking antibodies have been characterised in the art, including mAbs IE1, 2.2. 7.5 and 1 1 1.4 referred to in the Examples. As discussed above, blocking antibodies can conveniently be identified and/or characterised using assays that test their effect on neutralising antibody function. Modifications that may be made to produce MSP-1 variants of the invention include substitutions, deletions and insertions. It is particularly preferred to use substitutions to minimise disruption of the secondary/tertiary structure of the polypeptide. Furthermore, particularly preferred substitutions are those that replace one class of amino acid with another class, such as an aliphatic non-polar residue with a charged polar residue. For example, the twenty naturally occurring amino acids may be divided into four main groups (aliphatic non-polar [G, A, P, I, L and V], polar un-charged [C, S, T, M, N and Q], polar charged [D, E, K and R] and aromatic [H, F, W and Y]) and it is preferred to replace an amino acid from one group with an amino acid from another group. Other possibilities include replacing a positively charged side chain with a negatively charged side chain, replacing an amino acid with a large side chain with an amino acid with a smaller or no side chain (giycine), replacing a polar amino acid with a charged polar amino acid, replacing a large aromatic amino acid with an amino acid with a small side chain, and replacing cysteine residues that are involved in disulphide bonds. Particularly preferred modifications are an amino acid modification at any one of amino acid residues 14, 15, 27, 31, 34 , 43, 48 and 53 of the Plasmodium falciparum MSP-1 !9 amino acid sequence shown as SEQ I.D. No. 1 or their equivalent positions in other Plasmodium MSP-1 19 polypeptides. These residues are all almost within the EGF-like domain 1. It is known that the epitopes of some antibodies contain amino acid sequences that are within EGF-like domain 2. therefore equivalent modifications may also be made in EGF-like domain 2. Preferred examples of modifications include the following substitutions Glnl4→Arg, Glnl4→Gly, Asnl5- Arg, Glu27→Tyr, Leu31→Arg, Tyr34- Ser, Tyr34→Ile, Glu43— Leu. Thr48- Lys and/or Asn53→Arg and their equivalents in other Plasmodium MSP-1 1 9 polypeptides. It is especially preferred to carry out more than one modification, i.e. to use combinations of modifications, such as two or more or three or more. In a preferred embodiment, an MSP-1 variant of the invention comprises a combination of amino acid substitutions selected from [Glu27→Tyr. Leu31→Arg and Glu43— »Leu]. [Glu27- Tyr, Leu31-»Arg, Tyr34→Ser and Glu43→Leu], [Asnl5→Arg, Glu27→Tyr, Leu31→Arg and Glu43→Leu] and their equivalents in other Plasmodium MSP-1 !9 polypeptides. A particularly preferred combination further comprises a modification to Cysl2 and/or Cys28 (and/or their equivalent residues in EGF-like domain 2) to disrupt the disulphide bond. Preferably such modifications are substitutions selected from Cysl2— <-Ile and Cys28→Tφ, and Cysl2-→Ala and Cys28→Phe. Most preferably the substitutions are combinations selected from [Cysl2—*Ile. Asn 15→Arg, Glu27→Tyr, Cys28→Tφ, Leu31-→Arg, Glu43→Leu], [Cysl2→Ile, Asn 15→Arg, Glu27-→Tyr, Cys28→Tφ, Leu31→Arg, Glu43→Leu, Asn53→Arg], [Cysl2→Ile, Asn 15-→Arg, Glu27->Tyr, Cys28→Tφ, Leu31→Arg, Tyr34→Ser, Glu43— >Leu, Asn53-→Arg] and their equivalents in other Plasmodium MSP-1 1 polypeptides. Substitutions are not confined to using naturally occurring amino acids - non-naturally occurring amino acid analogues may also be used, in particular where solid phase synthesis is to be used to chemically synthesise the variant, as opposed to recombinant technology. Modifications to MSP-1 amino acid sequences may be carried out using standard techniques such as site-directed mutagenesis using the polymerase chain reaction. Alternatively, variants may be obtained by solid phase synthetic techniques. To determine whether a variant MSP-1 polypeptide produced by modification of its primary amino acid sequence complies with the criteria specified above, the affinity of at least one neutralising antibody and at least one blocking antibody for the variant polypeptide compared with the naturally occurring MSP-1 sequence may be tested. Ideally more than one of each type of antibody should be used, for example two or three. The ability of antibodies to bind to the variant and wild-type polypeptides may be determined using any one of a variety of methods available in the art for determining antibody-epitope binding. One such method, described in the Examples, involves the use of MSP-1 sequences expressed as fusion proteins with a protein tag such as glutathione-S- transferase (GST). These GST-fusion proteins are typically immobilised to a solid phase such as glutathione sepharose beads or a BIAcore sensor chip. Binding of antibodies, such as monoclonal antibodies, to the fusion proteins may be determined using standard techniques such as Western blotting and/or by labelling the antibodies with a radioactive label such as 12D I. The use of BIAcore technology allows easy quantitation of the results. Preferably, the reduction in binding of at least one of the blocking antibodies tested is at least 50% compared to wild-type MSP-1, more preferably at least 75, 80 or 90%, typically as assessed using recombinantly expressed MSP-1 immobilised to a BIAcore sensor chip. By contrast, the binding of at least one, for example at least two or three, of the neutralising antibodies tested, more preferably at least half of the neutralising antibodies tested, more preferably substantially all of the neutralising antibodies tested is reduced by less than 50%. more preferably less than 25%. The number of neutralising antibodies that need be tested to confirm compliance with the test criteria will not typically exceed from three to five different antibodies (three antibodies are used in the Examples). In a particularly preferred embodiment the binding of at least one neutralising antibody is increased by at least 10%. The results given in Table 2 in the Examples provide partial guidance to the skilled person as to which residues may be modified to produce a variant MSP-1 of the invention. However, the provision herein for the first time of the three dimensional solution structure of MSP-1 19 provides the skilled person with further detailed guidance as to which residues may be altered. In particular, epitopes are expected to be exposed to the aqueous environment on the exterior of the MSP-119 fragment. Consequently, the precise structural information provided which teaches the position of surface exposed amino acids allows the skilled person to target those residues for modification. This data is given in Tables A/B and has also been submitted to the Protein Data Bank (PDB Accession no. 1CEJ). It enables the skilled person to identify the precise location of individual amino acids in the three dimensional structure. Typically, the data is loaded into suitable software, well-known in the art such as Insight II. MOLSCRIPT GRAS P and RASMOL. Further, knowing the location of a modification in the 3-dimensional structure which affects the binding of a blocking antibody without affecting the binding of the neutralising antibodies, it is possible to identify other residues that are on the surface and in the vicinity of the original modification and which may be easily modified to further improve the properties of a modified protein. These residues may be in either the first or the second EGF-like motifs or in the sequence between them. Since it is known that an antibody binding site can encompass a volume that corresponds approximately to the range of 5 to 8 amino acids, it is clear that modifications of these adjacent residues may also affect the affinity of the protein for the blocking antibodies. Once an adjacent amino acid has been identified it can be modified according to the principles outlined above and the contribution of the modification to the overall antigenicity and immunogenicity of the protein, either alone or in combination with other modifications, can be assessed. Those changes that contribute to a reduced affinity for the blocking antibodies, without a substantial affect on binding of the neutralising antibodies can be incoφorated into the improved protein. This can be a reiterative process. In addition, the 3D NMR structure will enable the skilled person to carry out preliminary computer modelling studies of MSP-1 19 variants with specific modifications so that, for example variants that cannot fold properly may be discarded. This will assist in minimising the number of candidate MSP-1 19 variants that need be tested. Thus the present invention also provides a computer readable medium having stored thereon a model of the MSP-1 19 NMR structure. In a preferred embodiment, said model is built from all or some of the NMR data shown in Tables A and B. Variants of the present invention may optionally include additional MSP- 1 sequences, in particular regions of the MSP-I 33 region of MSP-1 2 to confer additional immunogenicity to the variant. Furthermore, additional sequences " known to contain and promote T cell responses are advantageously included (i.e. T cell epitopes). Other modifications may also be made that increase immunogenicity such as modifications that alter the pathway of antigen processing and presentation. Polypeptide variants of the invention are typically made by recombinant means, for example as described below. However they may also be made by synthetic means using techniques well known to skilled persons such as solid phase synthesis. Proteins of the invention may also be produced as fusion proteins-, for example to aid in extraction and purification. Examples of fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and/or transcriptional activation domains) and β-galactosidase. It may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein sequence of interest to allow removal of fusion protein sequences. Preferably the fusion protein will not hinder the immunogenicity of the MSP-1 variant. Polypeptides of the invention may be in a substantially isolated form. It will be understood that the polypeptide may be mixed with carriers or diluents which will not interfere with the intended puφose of the polypeptide and still be regarded as substantially isolated. A polypeptide of the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 90%o, e.g. 95%o, 98% or 99% of the polypeptide in the preparation is a polypeptide of the invention. B. Polvnucleotides and vectors As discussed above, the variants of the present invention may be produced recombinantly using standard techniques. Thus, the present invention also provides a polynucleotide encoding a polypeptide MSP-1 variant of the invention. Polvnucleotides of the invention may comprise DNA or RNA. They may. also be polvnucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the puφoses of the present invention, it is to be understood that the polynucleotides described herein may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of the invention. It will be understood by a skilled person that numerous different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code. Polynucleotides of the invention comprise can be incoφorated into a recombinant replicable vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention provides a method of making polynucleotides of the invention by introducing a polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell. Suitable host cells include bacteria such as E. coli, yeast, mammalian cell lines and other eukaryotic cell lines, for example insect Sf9 cells. The host cell may be a methylotrophic yeast such as Pichia pastoris. The coding sequence of natural or variant MSP polypeptides (including the polypeptide of the invention) may be modified for optimal expression in a host cell. For example, secondary modification such as N-glycosylation may be prevented by removal of sequences necessary for such modification. The sequence of the polypeptide may alternatively or in addition be modified with respect to codon usage for optimal expression in the host cell. Methods of mutagenising a sequence are known in the art; alternatively, the modified coding sequence may be generated by means of PCR gene assembly using overlapping synthetic oligonucleotides (Stemmer et al., 1995; Withers-Martinez et al.. 1999). Preferably, a polynucleotide of the invention in a vector is operably linked to a regulatory sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The term 'Operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is hgated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences. Such vectors may be transformed or transfected into a suitable host cell using standard techniques above to provide for expression of a polypeptide of the invention. This process may comprise culturing a host cell transformed with an expression vector as descπbed above under conditions to provide for expression by the vector of a coding sequence encoding the polypeptides, and optionally recovering the expressed polypeptides. The vectors may be for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector. Vectors may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell. The vector may also be adapted to be used in vivo, for example in a method of gene therapy Promoters/enhancers and other expression regulation signals may be selected to be compatible with the host cell for which the expression vector is designed. For example, prokaryotic promoters may be used, in particular those suitable for use in E. coli strains (such as E coli HB101 or DH5 ). When expression of the polypeptides of the invention in earned out in mammalian cells, either in vitro or in vivo, mammalian promoters may be used. Tissue-specific promoters may also be used. Viral promoters may also be used, for example the Moloney muπne leukaemia virus long terminal repeat (MMLV LTR), the promoter rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human cytomegalovirus (CMV) IE promoter, heφes simplex virus promoters or adenovirus promoters. All these promoters are readilv available in the art. C. Administration The variant MSP-1 polypeptides of the present invention and nucleic acid molecules may be used to treat or prevent malaria in animals, specifically humans. The polypeptides of the invention may be administered by direct injection. Preferably the polypeptides are combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition. Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline. The composition may be formulated for parenteral. intramuscular, intravenous, subcutaneous, intraocular or transdeπnal administration. Typically, each polypeptide is administered at a dose of from 0.01 to 30 μg/kg body weight, preferably from 0.1 to 10 μg/kg, more preferably from 0.1 to 1 μg/kg body weight. It is also possible to use antibodies prepared using the polypeptides of the invention, as described below, in treating or preventing Plasmodium infection. Neutralising antibodies, or fragments thereof which retain specificity for Plasmodium antigens, can be administered in a similar manner to the polypeptides of the invention. The polynucleotides of the invention may be administered directly as a naked nucleic acid construct. When the expression cassette is administered as a naked nucleic acid, the amount of nucleic acid administered is typically in the range of from 1 μg to 10 mg, preferably from 100 μg to 1 mg. Uptake of naked nucleic acid constructs by mammalian cells is enhanced by several known transfection techniques for example those including the use of transfection agents. Example of these agents include cationic agents (for example calcium phosphate and DEAE-dextran) and lipofectants (for example lipofectam™ and transfectam™). Typically, nucleic acid constructs are mixed with the transfection agent to produce a composition. Alternatively. the polynucleotide may be administered as part of a nucleic acid vector, including a plasmid vector or viral vector, such " as a vaccinia virus vector. When the polynucleotide of the invention is delivered to cells by a viral vector of the invention, the amount of virus administered is in the range of from 10 J to 10 pfu, preferably from 10 2 to 10 pfu. more preferably from 10 to 10 pfu. When injected, typically 1-10 μl of virus in a pharmaceutically acceptable suitable carrier or diluent is administered. Preferably the delivery vehicle (i.e. naked nucleic acid construct or viral vector comprising the polynucleotide for example) is combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition. Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline. The composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration. The routes of administration and dosages described are intended only as a guide since a skilled practitioner will be able to determine readily the optimum route of administration and dosage for any particular patient and condition. P. Preparation of Vaccines Vaccines may be prepared from one or more polypeptides of the invention. They may also include one or more immunogenic Plasmodium polypeptides known in the art. Thus a vaccine of the invention may comprise one or more polypeptides of the invention and optionally, one or more polypeptides selected from, for example, the asexual blood stage proteins: apical merozoite antigen- 1, erythrocyte binding antigen 175, erythrocyte membrane protein- 1 ; the hepatic stage proteins: liver stage antigens 1 and 3; the sporozoite stage proteins: circumsporozoite protein , thrombospondin related adhesive protein; and the sexual stage proteins Pfs25 and Pfs28 polypeptides and immunogenic fragments thereof. Preferably, the other immunogenic Plasmodium polypeptides known in the art do not contain wild type MSP-11 sequences. The preparation of vaccines which contain an immunogenic polypeptide(s) as active ingredient(s), is known to one skilled in the art. Typically, such vaccines are prepared as injectables. either as liquid solutions or suspensions: solid forms suitable for solution in. or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified, or the protein encapsulated in liposomes. The active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired. the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. Examples of adjuvants which may be effective include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-( -2'-dipalmitoyl-sn- glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835 A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. The effectiveness of an adjuvant may be determined by measuring the amount of antibodies directed against an immunogenic polypeptide containing an MSP-1 antigenic sequence resulting from administration of this polypeptide in vaccines which are also comprised of the various adjuvants. The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%>, preferably 1% to 2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25%) to 10%. Where the vaccine composition is lyophilised. the lyophilised material may be reconstituted prior to administration, e.g. as a suspension. Reconstitution is preferably effected in buffer. Capsules, tablets and pills for oral administration to a patient may be provided with an enteric coating comprising, for example, Eudragit "S", Eudragit "L". cellulose acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose. The polypeptides of the invention may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric and maleic. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethyiamine, 2-ethylamino ethanol, histidine and procaine. E. Dosage and Administration of Vaccines The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective. The quantity to be administered, which is generally in the range of 5 μg to 250 μg of antigen per dose, depends on the subject to be treated, capacity of the subject's immune system to synthesise antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered may depend on the judgement of the practitioner and may be peculiar to each subject. The vaccine may be given in a single dose schedule, or preferably in a multiple dose schedule. A multiple dose schedule is one in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the immune response, for example, at 1 to 4 months for a second dose, and if needed, a subsequent dose(s) after several months. The dosage regimen will also, at least in part, be determined by the need of the individual and be dependent upon the judgement of the practitioner. In addition, the vaccine containing the immunogenic MSP-1 antigen(s) may be administered in conjunction with other immunoregulatory agents, for example, immunoglobulins. F. Preparation of antibodies against the polypeptides of the invention The variant MSP-1 polypeptides prepared as described above can be used to produce antibodies, both polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunised with an immunogenic polypeptide bearing an MSP-1 epitope(s). Serum from the immunised animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to an MSP-1 epitope contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffmity chromatography. Techniques for producing and processing polyclonal antisera are known in the art. Monoclonal antibodies directed against MSP-1 epitopes in the polypeptides of the invention can also be readily produced by one skilled in the art. The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal antibody- producing cell lines can be created by cell fusion, and also by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. Panels of monoclonal antibodies produced against MSP-1 epitopes can be screened for various properties; i.e., for isotype and epitope affinity. The polypeptides of the invention can also be used to select for human monoclonal antibodies using the variable regions of immuno globulin heavy and light chains cloned in the form of a phage display library, preferably from individuals who have been previously exposed to a natural malaria infection. Antibodies, both monoclonal and polyclonal, which are directed against MSP-1 epitopes are particularly useful in diagnosis, and those which are neutralising are useful in passive immunotherapy. Monoclonal antibodies, in particular, may be used to raise anti-idiotype antibodies. Anti-idiotype antibodies are immunoglobulins which carry an "internal image " ' of the antigen of the infectious agent against which protection is desired. Techniques for raising anti-idiotype antibodies are known in the art. These anti-idiotype antibodies may also be useful for treatment of Plasmodium infections, as well as for an elucidation of the immunogenic regions of MSP-1 antigens. It is also possible to use fragments of the antibodies described above, for example, F(ab ' ) 2 , Fab, Facb and scFv fragments. It should be appeciated that features from various sections, aspects and embodiments of the invention as described above are generally equally applicable to other sections, aspects and embodiments mutatis mutandis. The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention. The Examples refer to the Figures. In the Figures: Detailed Description of the Figures Figure 1 - MSP-1 sequences aligned according to the EGF-like motif consensus. Top sequence: P. falciparum (SWISS-PROT MSP1 PLAFW). Second sequence: P. vivax Belem strain (PIR A45604). Third sequence: human EGF (PDB legf). Fourth sequence: EGF-like domain consensus (Prosite EGFl). Bottom sequence: 14 residue EGF core region used for structure alignment in Figure 6. Black highlighting indicates conserved residues of the EGF-like domain. Dark shading shows hydrophobic residues at the EGF- module pair interface in the P. falciparum, and corresponding conserved residues in the P. vivax sequence. Figure 2 - Sample of multidimensional heteronuclear NOESY experiments showing planes containing NOE connections to the MSP-1 C-terminal fragment Lys35 NH proton. Top: 13 C (D4) and Η(D3) plane from the 4D-[ 13 C]-HMQC-NOESY-[ 15 N]-HSQC experiment, taken at the chemical shift values of Lys35 NH in ! "N(D2) and Η(D1). Bottom: strip from the 3D [ 15 N]-NOESY-HSQC at the Η chemical shift value of Lys35 NH (vertical axis, Dl) taken at the plane of its ' " N (D3) value. The horizontal 'H axis is aligned with that of the top spectrum. The weak cross-peaks at 2.72 and 3.01 ppm in the 3D spectrum do not show corresponding cross-peaks in the 4D spectrum because of the lower signal-to-noise ratio in the latter. These peaks have been assigned as the cross-peaks between Lys35 NH and Asn44 H β2 (2.72 ppm), and Cys30 H β3 and/or Cys41 H β2 (3.01 ppm). Figure 3 - Stereo drawing showing the backbone C, N, C a atoms of the 32 refined structures in the final ensemble. The domain- 1 is on the left (red), with domain-2 on the right (green), and both the N- and C-termini are near the bottom. Figure 4 - MOLSCRIPT picture of the most representative model of the ensemble, showing the backbone C α trace, antiparallel β-sheet elements, and disulphide bridges (S γ atoms in yellow). Domain- 1, red; Domain-2, green. Figure 5 - Alignment of typical EGF-like family members with the fitpdb program, using the 14 amino acid "reduced core" consensus (Bersch et al. 1998) (see Figure 1). The aligned backbone segment in each structure is white. The structures are aligned relative to the most representative structure of the group (factor Xa), with increasing divergence from left to right. Numbers indicate the rmsd value of the aligned C, N, C α atoms. PDB identification codes: factor Xa (crystal structure), lhcg; Complement Clr component, lapq (14 th model); human EGF, legf (11 th model); fibrillin-1, domains-32 and -33 ^ 1emn (minimized average structure); transforming growth factor-α, 2tgf (minimized average structure); MSP-1 domains- 1 and -2, this study. Figure 6 - Backbone ribbon view of fibrillin-1 versus MSP-1 EGF module pair arrangements. Fibrillin-1 (lemn) cyan (domain-32) and magenta (domain-33) (Downing et al, 1996): MSP-1 domain- 1 (yellow) and domain-2 (green). Structures were aligned as in Figure 6 by the core consensus of the N^terminal domain of each pair. The bound Ca "" ions in the fibrillin-1 structure are shown as magenta spheres. Figure 7 - Two views, a and b. (rotated 180° about-the y-axis) of the electrostatic potential surface of the MSP-1 EGF module pair, calculated with GRASP. Red indicates negative charge, blue indicates positive charge, and white is neutral. The orientation of the views is shown by the adjacent worm diagrams. Figure 8 - CPK model of the MSP-1 C-terminal fragment, showing the location of some mutations that affect binding of monoclonal antibodies. Domain- 1 is towards the top and right sides, and domain-2 towards the bottom left. Figure 9 - Examples of the binding of monoclonal antibodies to GST-MSP-1 19 detected by Western blotting. The binding of each monoclonal antibody to protein based on the wild type sequence and to proteins containing modified sequences is shown. The monoclonal antibodies are shown across the top. On the left is shown the proteins: WT, wild type sequence; 22, Leu22 to Arg; 26, Glu26 to He; 15, Asnl5 to Arg; 27, Glu27 to Tyr; 31, Leu31 to Arg; 43, Glu43 to Leu; 27+31+43, Glu27 to Tyr and Leu31 to Arg and Glu43 to Leu; 15+27+31+43. Asnl5 to Arg and Glu27 to Tyr and Leu31 to Arg and Glu43 to Leu. Figure 10 - The binding of monoclonal antibodies to GST-MSP-1 1 detected by BIAcore analysis. The binding of each monoclonal antibody is normalised to 100% binding to protein based on the wild type sequence and the binding of proteins containing modified sequences is expressed as a percentage of this. WT, wild type sequence; 15, Asn 15-→Arg; 26, Glu26→Ile; 27, Glu27→Tyr; 31, Leu31-→Arg; 34, Tyr34-→Ser; 43 Glu43→Leu. Figure 11 - The binding of monoclonal antibodies to GST-MSP-1 19 containing multiple modifications detected by BIAcore analysis. The binding of each monoclonal antibody is normalised to 100%) binding to protein based on the wild type sequence and the binding of proteins containing modified sequences is expressed as a percentage of this. WT, wild type sequence; The combinations contain 3 mutations [27+31+43], or 4 mutations ([27+31+34+43] and [15+27+31+43]). at each site the changes are those identified in Figure 10. Figure 12 - Identification of blocking antibodies using a competitive binding assay and immobilised wild type GST-MSP-1 19 The ability ' of antibodies to compete with the binding of mAbs 12.8 and 12.10 to GST-MSP-1 19 was measured using BIAcore analysis. Individual antibodies (x-axis) were bound to the antigen and then the amount of either 12.8 or 12.10 (inhibitory mAb) that could subsequently bind was quantified. The amount of binding is presented as a percentage of the total amount of either 12.8 or 12.10 bound in the absence of pre-incubation with another antibody. Figure 13 - Antibodies induced by immunisation with a modified recombinant MSP-1 19 assayed for their ability to inhibit secondary processing. Washed 3D7 merozoites were either analysed directly without incubation (0 h) or incubated for 1 hour at 37°C in the presence of no serum (no serum), 1 mM PMSF as a control for complete inhibition, normal rabbit sera (normal serum), or serum from a rabbit immunised with the 15+27+31+43 modified protein (immune serum), all at 1 : 10 dilution in reaction buffer. The level of MSP-1 33 released into the supernatant as a results of secondary processing was measured using an ELISA method and is represented by Absorbance at 492nm. Figure 14. Pichia pastoris codon preference table used for input to the CODOP program. Figure 15. DNA and protein sequences for the optimized synthetic MSP-142 gene. A: Complete sequence designed for optimum codon usage and expression in P. pastoris. B: Sequence of the synthetic MSP-1 19 construct in the expression vector pPIC9K-HXa. Uppercase letters: vector sequences, including the His 6 tag and factor Xa cleavage site (IEGR). Lowercase letters: synthetic MSP-1 19 coding sequence. The cloned sequence in located at the SnaBI restriction site of the pPIC9K sequence. C: Expressed protein sequence of the synthetic MSP-1 19 construct. The sequence shown is produced as a fusion to the pPIC9K -factor secretion signal, following the kex2/STE13 processing sites. The synthetic MSP-119 is in bold-face type. D: Sequence of the MSP-133 construct. The cloned sequence is located at the Smal site of the pUCl lδ vector. E: Predicted protein sequence of the synthetic MSP-133 construct translation product. Figure 16. Gene assembly PCR reactions for the MSP-133 and MSP-119 sequences. Reaction 1 : 10 μL aliquots of the assembly reactions. Reaction 2: 20 μL aliquots of the amplification reactions. The N-terminal and middle fragments were subsequently spliced together to form the MSP-133 synthetic construct. The C-terminal fragment synthesis reactions produced the optimized MSP-1 19 construct. Figure 17. Expression of the synthetic MSP-1 19 protein in P. pastoris. Lanes 1-6: trichloroacetic acid precipitates of secreted recombinant protein from culture supematants, without further purification (5 μL each). Samples from duplicate cultures of three independent transformants. Lane 8,9: purified, deglycosylated MSP-119 produced from the original P. falciparum sequence. Lane 7,10: NOVEX molecular weight markers. Figure 18. A: {'H/ 1: N}-HSQC spectrum of the protein (2.5 mM) expressed from the optimized synthetic MSP-119 gene. B: Control {'H/ 15 N}-HSQC of deglycosylated protein (2.2 mM) expressed from the original P. falciparum sequence (Morgan et al., 1999). Examples Materials and Methods Protein expression and stable-isotope labelling for NMR The coding sequence of the MSP-1 C-terminal fragment was cloned by polymerase chain reaction with Vent polymerase (New England Biolabs) from a plasmid containing the Plasmodium falciparum strain T9/94 fragment (Blackman et al, 1991), using primers that included codons for a 6 residue N-terminal His tag (CACCATCATCATCATCAC), and inserted into the SnaBI restriction site of the pPIC9K vector (Invitrogen). The sequence corresponds to residues 1526-1621 of the SWISS-PROT entry MSP1 PLAFW (accession number P04933). This produced an α-factor fusion protein with the sequence ...KR/EA/EA/YHHHHHHNISQ....SSSN. where the slashes indicate kex2 and STE13 processing sites. High copy number transformants of the methylotrophic yeast Komagataella (Pichia) pastoris protease-deficient strain SMD1168 (his4 pep4) were isolated by screening for high G418 resistance (Clare ei al, 1995). A Mut " transformant was grown at 29.4 °C in a shaker-incubator in buffered minimal medium ( 100 mM potassium phosphate. pH 6.0. yeast nitrogen base (0.34 % w/vol) (DIFCO: YNB without amino acids and without (NH )2S0 ). biotin (4x10°% w/vol), Sigma antifoam 289 (0.01% vol/vol). and carbon and nitrogen sources as described below. Unlabelled samples were initially grown in medium containing 1 % w/vol (NH 4 ) 2 S0 4 and 1 % w/vol glycerol. and induced by transfer to medium containing 0.5 % CH 3 OH as the carbon source. Labelled samples were initially grown in medium containing 0.2 % w/vol [ 15 N]-(NH ) 2 S0 4 (Isotech), and 0.5 % w/vol glucose or [ 13 C 6 ]-glucose (Isotech), and induced by transfer to medium containing as carbon source 0.5 % w/vol CH 3 OH or [ lj C]- CH 3 OH (Isotech). The initial cultures were grown in 150 ml to a density of -10 ODβoo, then harvested and resuspended in methanol medium at 1 OD 6 oo in a volume of 1.5 L. Methanol-induced cultures were grown for 4 d. with daily addition of 7.5 ml CH 3 OH or [ lj C]-CH 3 OH, to a final density of -18 OD 6 oo- This protocol produced a maximum yield of 24 mg/L of purified, lj C/'"N uniformly labelled protein at the final stage (see below). The YNB-based medium produced about 3-fold higher yields than the FM22 medium (Laroche et al, 1994), for stable-isotope labeling of the MSP-1 C-terminal fragment. Cells were removed by low-speed centrifugation, protease inhibitors added (COMPLETE™ tablets. Boehringer-Mannheim: 1 tablet/500 ml supernatant), and the supernatant was filter-sterilized. The supernatant was concentrated -20-fold by ultrafiltration in a stirred cell (Amicon. YM3 membrane) at 4 °C. The pH was adjusted to 7.25 with KOH, and the partially N-glycosylated MSP-1 fragment was deglycosylated for 72 h at 37 °C with 5000 U PNGaseF (New England Biolabs). The carbohydrate was completely removed (as shown by electrophoresis and mass spectrometry), with the Asn 1 residue presumably converted to Asp in the process. The supernatant was clarified _by low- speed centrifugation, 5 M NaCl was added to a final concentration of 0.3 M. and the sample was applied to a 2 ml Ni-NTA affinity column (QLAGEN), washed, and eluted with 250 mM imidazole according to manufacturer's instructions. The eluate was dialyzed against 50 mM sodium phosphate (pH 6.5), 50 mM NaCl, and then passed through a 1 ml Hi-Trap Q anion exchange resin (Pharmacia) to remove misfolded MSP-1 that bound to the column. The MSP-1 fragment was characterized by Western blotting and electrospray mass spectrometry (data not shown). Two principal species of mass 11607 and 1 1807 Da were observed corresponding to the expected fragment, as well as a fragment with an additional N-terminal Glu - Ala dipeptide resulting from incomplete STE13 processing of the α-factor secretion signal. " Samples for NMR experiments were prepared in either 90 % H 2 O/10 % D 2 0 with 0.01 % w/vol NaN 3. or 100 % D 2 0, 50 mM sodium phosphate, 100 mM NaCl at pH 6.5, (pH uncorrected for deuterium isotope effects), at a concentration of 2.1 to 2.6 mM in 0.6 ml. Protein concentration was measured by UV absorbance at 280 nm, using a calculated molar extinction coefficient of 5220 liter mol -1 cm -1 . The protein was demonstrated to be monomeric by equilibrium ultracentrifugation of a 0.12 mM sample in the above buffer at 293 K. NMR experiments and data processing Most of the experiments were performed at 298 K, using Varian Unity and Unity-Plus spectrometers operating at 600 MHz and 500 MHz respectively. Details of the multidimensional experiments (Clore & Gronenborn, 1998) and acquisition parameters used for resonance assignments and structure determination are given in Tables A/B and have been submitted to the Protein Data Bank database (PDB Accession No 1CEJ). All spectra were processed using Felix 95.0 or 97.0 (Biosym/MSI) using a 90 degree- or 72 degree-shifted sinebell-squared window function. Dimensions, zero-filling, and linear prediction details are summarized in Tables A/B and in the submission to the BioMagResBank. Four dimensional and interleaved spectra were processed in Felix using macros written in-house. Signal assignments: Sequential assignments were made based on connectivities established primarily by CBCA(CO)NH and CBCANH experiments on uniformly 13 C/ 15 N labelled protein. Side chain-spin system assignments were made on the basis of data from 13 C/'H -HCCH-TOCSY experiment correlated with information from 15 N/Η-TOCSY- HSQC and I5 N/Η-NOESY-HSQC. and FINHA and HNHB experiments. Assignments were obtained for Η, l 5 N and aliphatic - lj C signals for 98%) of side-chains and 96%> of backbone amide groups. The list of assignments is given in Tables A B and in the submission to the Protein Data Bank database (PDB Accession No 1CEJ). The '"N{Η} heteronuclear NOE experiment was carried out as " described previously (Kay et al, 1989; Polshakov et α/., 1997). Distance Restraints: NOE- and ROE-derived distance restraints between backbone and side chain amide protons were obtained primarily from the 3D '"N-NOESY-HSQC, 15 N-ROESY-HSQC, and 4D 13 C-HMQC-NOESY- 15 N-HSQC experiments. Aliphatic to aliphatic proton distance restraints were obtained from a 4D lj C-HMQC-NOESY- lj C- HSQC experiment. A 3D 13 C-HMQC-NOESY experiment in D 2 0 was used to identify aliphatic to aromatic proton NOEs and 2D NOESY experiments were used to measure aromatic to aromatic proton NOEs. Crosspeaks were quantified by volume integration in Felix for 2D and 3D experiments and for the 4D I3 C-HMQC-NOESY- 15 N-HSQC experiment, and from peak height measurements in the 4D 13 C-HMQC-NOESY- 13 C- HMQC spectra. Crosspeaks were classified as strong, medium and weak and these were assigned to distance restraints of 0 - 2.8, 0 - 3.6, and 0 - 5.5 A. Restraints from backbone amide signals were initially treated in this manner, and then recalibrated more precisely using 3D- l: N-ROESY-HSQC data into four classes involving maximum distances of 2.6, 3.1, 3.6, and 4.1 A. Restraints to groups of equivalent or non-stereoassigned protons were treated by r " summation. Most intraresidue distances (HN-Hβ and H α -H β ) were converted to χi angle restraints as described below and these distance restraints were not included in the final list. Dihedral Angle Restraints: χ angles and stereospecific assignments of β-methylene protons were obtained using the grid-search program AngleSearch, with coupling constant and intraresidue ROE distance information (Polshakov et al, 1995). The coupling constant information was provided by HNHB and HN(CO)HB spectral intensities for J J(HN-H β ) and J J(CO-H β ), and intraresidue distances (HN-H β , H α -H β ) were obtained from 3D 15 N-ROESY-HSQC and 2D ROESY (D 2 0) experiments. 3 J(HN-H α ) coupling constants were obtained from the HNHA experiment. Residues with positive φ angles (ca. -60 degrees) were identified by large intraresidue H α crosspeak intensities in the HN(CO)HB experiment, and y angles near -60° degrees from strong H α (,.π crosspeaks in the HNHB experiment. lie and Leu χ 2 angles and Leu δ stereoassignments were derived from the LRCH experiment. Minimum ranges of 40 degrees (χi , χ ) and 50 degrees (φ. ψ) were used to account for errors and local dynamic effects on the coupling constants. Disulphide Bonding Pattern: An initial set of 20 structures was calculated by simulated annealing using approximately 550 unambiguous NOE-derived distance restraints and 36 χi and φ dihedral angle restraints but with no hydrogen bonding or disulphide bond constraints. The Cys - Cys S γ distances in these structures were examined in order to establish the probable bonding pattern. Prior to the calculations, the formation of disulphide bridges for 4 Cys residues (Cys 12 - Cys28, Cys78 - Cys92) was already established with high probability by the observation of H β -H NOEs between these pairs of Cys residues. Examination of the initial structures confirmed these disulphide bridges and also indicated a disulphide bridge between residues Cys30 and Cys41. The third disulphide bridge in domain- 1 (Cys7 - Cys 18) could thus be assigned by default, although the structure of the N-terminus was not well-defined by the NMR data. The best six structures in terms of total X-PLOR energy and violations indicated that the average Cys - Cys Sγ distance was lowest for the disulphide bonding pattern [1-3, 2-4, 5-6] in each domain, and only this combination allowed all Cys residues to form contacts with a partner <3.5 A away. Thus, this disulphide bonding pattern was most consistent with the experimental data for both domains, and was imposed (initially as NOE-style distance restraints) in subsequent calculations. The [1-3, 2-4, 5-6] pattern is that expected for an EGF-like domain. Hydrogen Bonds: Non-exchanging amide groups involved in stable hydrogen bonds were identified in spectra of samples examined in 100 % D 2 0. The corresponding hydrogen bond acceptors were determined by examining the initial structural ensemble, using the Insight II and HBPlus (McDonald et al, 1994) programs, and hydrogen bond distance restraints were included in subsequent calculations. Further hydrogen bonds were identified in a similar manner in iterative calculations. Only 10 backbone hydrogen bonds in the antiparallel β sheets were used as restraints. Two distance restraints were used for each hydrogen bond, 1.7 - 2.3 A from proton to acceptor, and 3.0 - 3.6 A from donor nitrogen atom to acceptor. Structure Calculations All the structure calculations were performed following standard protocols for ab initio simulated annealing from an extended chain using X-PLOR version 3.843 on a Silicon Graphics Origin 200 computer. The initial calculations used an initial temperature of 1000K. and 9000 steps of 5 fs in the restrained molecular dynamics stage. A soft-square potential was used for distance restraints. The SHAKE (Ryckaert et al, 1977) algorithm was employed during molecular dynamics to maintain correct bond lengths. Refinement used a square well potential for restraints, and a final slow cooling of 30000 steps of 4 fs each from 2000K. A modified "parallhdg.pro" force-field parameter set was used, with modifications to parameters for Arg and Pro residues, and for hydrogen bonds (Polshakov et al. 1997). Force constants were 50 kcal mol "1 A "" for all distance restraints including hydrogen bonds, and 200 kcal mol "1 rad "2 for dihedral restraints. The N-terminal sequence including the vector-encoded residues and (His) 6 tag was excluded from the structure calculations. All peptide bonds were constrained to be trans. NOE data for all 5 Pro residues showed strong H α (,.i)-ProH α crosspeaks, consistent with the trans peptide conformation. Initial structures were calculated as described above to determine the disulphide bonding pattern. Then the calculation was repeated with identical NOE-derived distance and dihedral angle restraints, with the addition of 6 distance restraints (1.92-3.12 A) representing the disulphide bridges. A new set of 50 structures was obtained, from which the best 20 structures were selected. The criteria used for selection were that the structures were below the median value of both total X-PLOR energy and rms NOE difference, and had no dihedral angle violations. The resulting structures had good geometry and between zero and two NOE violations > 0.5 A. These structures were used to assign previously ambiguous NOEs and to determine the hydrogen bonds as described above. The final structure calculation and refinement used an expanded restraint list including hydrogen bonds, additional dihedral restraints, stereoassignments of β-methylene and Leu δ signals, and more precisely calibrated ROE data (see Table 1). A set of 100 structures was obtained using this list, and 38 structures with 0-2 NOE violations > 0.5 A and no dihedral angle violations > 5° were accepted. These 38 structures were refined by the slow-cooling procedure described above, producing a final ensemble of 32 accepted structures with no NOE violations > 0.5 A and no dihedral angle violations > 5°. These selection criteria produced an ensemble of structures that extend to the end of the continuum of total potential energies in order to include structures having large scale correlated motions (Abseher et al, 1998). Statistics for the final ensemble are given in Table 1. Coordinates for the 32 refined structures have been deposited in the Brookhaven Protein Data Bank (coordinates ID code lcej; NMR restraints ID code rlcejmr). Structures were analyzed during the calculation process using X-PLOR 3.8 (Nilges et al, 1991), PROCHECK-NMR/AQUA (Laskowski et al, 1996), and Insight II for quality of agreement with experimental data, precision, geometry, and energy. Models were aligned with Insight II and fitpdb. and displayed with Insight II, MOLSCRIPT (Kraulis, 1991), and GRASP (Nicholls et α/.,1991). Table 1. A: RESTRAINTS SUMMARY Number of conformers calculated: 100 Number of conformers accepted: 32 Acceptance criteria: No distance violation: > 0.5 A No dihedral angle violation: > 5 ° NOE/ROE distance restraints: Intraresidue: 73 Sequential: 222 Medium range ( 2-4 ): 90 Long range ( > 4 ): 185 Total: 570 Dihedral angle restraints: phi: 25 psi: 33 chi-l : 22 - chi-2: 5 Total: 85 Hydrogen bonds: 10 Disulphide bonds: 6 B: STRUCTURE QUALITY average -/- s.d. Total X-PLOR energy (kcal mol "1 ) 168 20 NOE X-PLOR energy (kcal mol "1 ) 21 rmsd NOE 0.026 0.005 rmsd dihedral angle 0.236 0.095 rmsd bond length 0.0029 0.0002 rmsd bond angle 0.357 0.023 rmsd improper 0.266 0.018 Backbone rmsd of structured regionn: (69 residues) Overall: 1.05 0.28 Domain- 1: 0.81 0.32 Domain-2: 0.83 0.35 Ramachandran plot quality (phi psi angles): Most favoured 49.5 % Additional allowed 42.1 % Generously allowed 5.6 % Disallowed 2.7 % Monoclonal antibodies (mAbs) Anti-MSP-1 19 monoclonal antibodies used in this study were : mouse IgG mAbs 1E1, 1E8. 2F10, 1 11.2, 111.4 2.2. 5.2, 7.5. 9C8, 12.8, 12.10, 12D11, 117.2, 8A12 (Holder et al. 1985; McBride & Heidrich, 1987; Blackman et al. 1987; Guevara Patino et al. 1997); and mouse IgM mAb 5B1 (Pirson & Perkins, 1985). Of these, mAbs 12.8, 12.10 and 5B1 are neutralising, inhibitory antibodies and 1E1, 2.2, 7.5, 9C8 and 111.4 are blocking antibodies. Some antibodies such as 1 1 1.2 are neither inhibitory nor blocking. The DNA coding the wild type MSP-1 19 domain of Plasmodium falciparum (T9- 94/Wellcome strain) MSP-1 has been cloned in expression vector pGEX-3X to produce MSP-1 19 fused to the carboxy-teπninus of the Schistosoma japonicum glutathione S- transferase (GST) in Escherichia coli (Burghaus & Holder, 1994). Site-directed mutagenesis of MSP-1 19 DNA sequence was done in either of two ways. The first method was a modification of the method of Perrin & GiUiland (1990) to carry out polymerase chain reaction (PCR)-mediated site specific mutagenesis. DNA was amplified using the plasmid as a template together with one oligonucleotide to introduce the point mutation and a 5' primer from outside of the MSP-1 19 sequence. The amplified product was purified after electrophoresis on an agarose gel and used in a second amplification step together with a 3' primer from outside of the other end of the MSP-1 19 sequence and the plasmid as template. This second PCR product was digested with the restriction enzymes EcoRl and BamRl and the product consisting of the modified MSP- l ι 9 coding sequence was inserted back into pGΕX-3X and the products were used to transform DH5α cells. The second method used the QuikChange™ Site-directed mutagenesis kit from Stratagene. Briefly, using the plasmid pGEX-MSP-l ι 9 as a template, two complementary synthetic oligonucleotide primers containing the desired point mutation were designed and were extended on the template by temperature cycling with the enzyme Pfu DNA Polymerase. This incoφoration of the oligonucleotide primers results in the generation of a mutated plasmid containing staggered nicks in the DNA sequence. Following the temperature cycling, the product was treated with Dpnl endonulease which will digest the methylated parental DNA template and leaves the mutation-containing newly synthesised DNA intact. The DNA incoφorating the desired mutation was then transformed into E. coli strain DH5α (Life technologies) competent cells where the nicks will be repaired. Clones were screened by analysis of restriction enzyme digests and by PCR screening of the insert gene. The DNA sequence of the selected mutant clones was confirmed using a PerkinElmer Applied Biosystems ABI 377 automatic sequencer according to the manufacturer ' s instructions. Expression of the GST-MSP-1 IQ fusion proteins Expression of GST-MSP-1 J9 was induced with 1 mM isopropyl-β-D- thiogalactopyranoside (IPTG; Melford Laboratories) for 1 hour in the E. coli strain TOPP 1 (Stratagene). The cells were then harvested by centrifugation and the cell pellet was resuspended in cell lysis buffer (50 mM Tris-HCl/1 mM EDTA pH 8.0 containing 0.2% (v/v) Nonidet P40 (NP40; BDH). Phenylmethylsulphonyl fluoride (PMSF; Sigma) in isopropanol was added to a final concentration of 1 mM. The cell suspension was sonicated, on ice, using VibraCell sonicator (Sonics & Materials) at 50%) duty cycle for 3 min (six 30 sec pulses with 30 sec in between). The cell lysate was centrifuged at 65000 x g for 1 hour at 4°C. Supernatant containing soluble GST-fusion protein was applied to a glutathione-agrose column (Sigma) and the GST-fusion protein was eluted with 5 mM reduced glutathione. The eluted GST-fusion protein was dialysed extensively against phosphate buffered saline (PBS) at 4°C. SDS-PAGE and Western Blotting Proteins were analysed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE). Samples were solubilised in SDS-PAGE buffer without reducing agents, then fractionated on a homogeneous 12.5% polyacrylamide gel. The pre- stained low range molecular mass markers (24-102kDa) from Bio-Rad were used as markers. When required, SDS-PAGE-fractionated polypeptides were either stained with Coomassie Brilliant Blue R-250 (CBB; Sigma) or electrophoretically transferred to Optitran BA-S 83 reinforced nitrocellulose (Schleicher & Schull, 0.2 μm pore size) for analysis by western blotting. Blots were blocked with 5% BSA. 0.5% Tween 20 in PBS (PBS-T) for 1 h at room temperature, then washed in PBS-T. Blots were probed with first antibodies for 2 h at room temperature, -washed 3 times in PBS-T, and then incubated in 1/1000 dilution of horse radish peroxidase (HRP)-conjugated sheep anti-mouse IgG (H-'-L) (ICN Immunobiologicals) or Goat anti-mouse IgM (μ chain) (Sigma) for 1 h at room temperature. Blots were then washed 3 times in FBS-T and developed using Super Signal Substrate (Pierce) as HRP substrate for 1 min. Blots were then placed in plastic wrap and exposed to X-ray film (XB-200. X-ograph Imaging Systems). The films were processed with an Agfa Gevamatic60 film processor (Agfa). Analysis of antibodv-antigen interaction using a BIAcore machine GST-MSP-119 containing either the wild type or various modified sequences was used to coat a carboxymethyl dextran hydrogen sensor chip by the following methodology. The binding of the GST-MSP-1 19 was via amino groups using EDC/NHS chemistry. Immobilisation was done with the amine coupling kit (Pharmacia BIAcore). The CM dextran surface was activated with 50 μl of 200 mM l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 5 mM N-hydroxysuccinamide (NHS) for 10 min. GST-MSP-1 19 was then coupled to the BIAcore sensor surface using 50 μl of a solution at 100 μg ml "1 in coating buffer (0.01M sodium acetate buffer, pH 3.5) for 10 min. Unreacted carboxyl groups were blocked by adding 50 μl 1 M ethanolamine, pH 8.5 for 10 min. The cells were washed with two pulses of 20 μl 10 mM glycine-HCl, pH 2.8 for 8 min in total to remove any non-covalently bound protein. The immobilisation procedure was carried out at a flow rate of 5 μl min "1 . Measurements were performed on the BIAcore 2000 instrument. RESULTS Example 1 - Resonance assignments, NMR restraints and structure determination The assignments and restraints were obtained as described in Materials and Methods using a range of multidimensional heteronuclear experiments with ' C/ 15 N uniformly labelled protein. Sample spectra from 3D and 4D experiments showing NOE connections to the Lys35 backbone amide NH proton, resolved and unambiguously assigned using the 13 C chemical shift information, are shown in Figure 2. The distance, dihedral angle and hvdrogen bond restraints used in the final set of structure calculations are summarized in Table 1. A total of 570 unambiguously assigned distance restraints, 85 dihedral angle restraints, and 10 hydrogen bonds were used in the final set. The assignments and restraint list shown in Table A have been submitted to the BioMagResBank database. Three disulphide bonds, with the (1-3. 2-4, 5-6) pattern for each domain were experimentally determined from the NMR data in preliminary calculations as described in Materials and Methods, and these were also included in the final refinement. A final set of 32 models was calculated and refined using these restraints and these structures are shown in Figure 3 superimposed on the backbone of the representative structure S rep . Table 1 shows that all 32 models have good geometry and are in good agreement with the experimental data with no NOE violations > 0.5 A and no dihedral angle violations > 5°. The atomic rmsd value for the backbone atoms of the well-structured region (residues 15 - 64, 74 - 92) is 1.05 A (see Table 1). The local backbone rmsd is highest at the N-terminus (up to Cysl2), in the loop Glu65 - Lys73. and following Cys92 at the C-terminus. The Ramachandran plot quality is typical of that found for other EGF structures (Doreleijers et al, 1998). Description of the structure EGF-domains Analysis of the final ensemble by PROCHECK-NMR indicated that each domain contains a major stretch of antiparallel β-sheet containing the third and fourth Cys residues of each domain, as expected for an EGF-like fold, as well as an additional minor antiparallel β-sheet at the C-terminal end of domain- 1, similar to some (but not all) EGF family members. These secondary structure features, together with the disulphide bonding patterns, can be seen in Figure 4. There is also a well-defined type II tight turn in domain- 1, with a hydrogen bond from Tyr 34NH proton to Leu31 carbonyl oxygen. The normally conserved EGF consensus Gly residue in the tight turn is replaced in domain- 1 by a residue with a positive φ angle (Asn33), while the conserved aromatic residue is present (Tyr34). There is a probable hydrogen bond between Leu 31NH proton to Asnl5 carbonyl oxygen. Domain-2 contains two turns preceding the major β-sheet, (Asn53 - Cys56, Asp57 - AlaόO), and a final bend from Leu86 - Phe91 with a probable hydrogen bond from Asp57 NH proton to the carbonyl oxygen of Ile90 or Gly89. A surface-exposed loop from Pro81 to Pro85 replaces the tight turn, while the aromatic residue is not conserved. The large loop at the end of the major b-sheet (Glu65 - Lys73) is relatively disordered, and high mobility for the segment Gly68 - Gly71 was confirmed by backbone amide lD N{Η} heteronuclear NOE measurements (Barbato et al. 1992). The heteronuclear NOE values are dramatically reduced for residues in this region. At the N-terminus: the low NOE intensities correspond to increased mobility compared with the rest of the protein. The interdomain linker region from Pro45 to Pro47 is distinct from other EGF-like module pairs. The conformations of the disulphide bridges between Cys30 - Cys41 in domain- 1, and the three Cys - Cys bonds in domain-2 are all left handed spirals (Richardson, 1981). Bridges between Cys30 - Cys41, Cys56 - Cys76, and Cys78 - Cys92 are particularly close to their equivalents in the blood coagulation factor Xa structure (lhcg). The conformations of the first two disulphide bridges in the relatively disordered N-terminal segment of domain- 1 were not determined. Figure 5 shows the backbone C, N, C α atom alignments of the two MSP-1 C-terminal fragment domains made with typical examples of EGF-like domains from several proteins, using the fitpdb program. Pairwise alignments showed that the two domains from MSP-1 are more similar to the factor Xa structure and its close relative from Clr, than to each other or to the other structures tested. The rmsd values for MSP-1 domains compared to factor Xa are comparable to those of the more distantly related structures fibrillin-1 and transforming growth factor-α. The overall fold of each MSP-1 domain is thus similar to typical EGF family members, with the turns following the fifth Cys residue roughly equivalent, in spite of the divergence from the EGF consensus (C(5)xxGα) where α is a Phe or Tyr -residue. Although some of the external loops are disordered, the scaffold is quite stable, as indicated by the non-exchangeable backbone amides (see above and in Protein Data Bank/BioMagResBank submission for details). Unlike many EGF-like domains such as fibrillin-1, the MSP-1 C-terminal fragment lacks the conserved EGF Ca" + -binding sequence and there was no evidence of Ca" + binding to the MSP-1 C-terminal fragment. The 2D Η-NOESY spectra were virtually identical in the absence or presence of 20 mM CaCl 2 , indicating that any binding that might occur has, at most, only a small affect the overall structure. Domain interface and surface The most striking feature of the MSP-1 C-terminal fragment structure is the interface between the domains, which consists of several nonpolar amino acids (Phel9. Leu31. Leu32. Leu86, Phe87, Ile90 and Phe91) involved in hydrophobic interactions. These residues join the base of the major β-sheet and the tight turn in domain-1 with the final bend from residue 86 to 91 in domain-2. The domain interactions result in the domains forming a U-shaped structure which contrasts with structures observed for other pairs of EGF domains (Downing et al. 1996; Brandstetter et al, 1995). For example, in fibrillin-1, the interface between EGF domains 32 and 33 is largely formed by a shared Ca 2+ ligation site (Downing et al. 1996), and the overall structure resembles a rigid rod, with distant N- and C-termini. This contrasts with MSP-1 where the EGF-like domains are folded against each other so that their termini are relatively close together. A comparison of fibrillin-1 and MSP-1 EGF module pairs is shown in Figure 6. Although both termini of the MSP-1 C-terminal fragment are somewhat disordered. NOE contacts were observed between nuclei in the two ends. The proximity of the C- and N-terminal positions may be significant, since it suggests that the proteolytic processing site that produces the C- terminal 96 amino acid fragment may be very close to the GPI membrane attachment site at or near residue 96. This proximity is consistent with the idea that a membrane-bound Plasmodium proteinase is responsible for secondary processing. The electrostatic potential surface of the MSP-1 C-terminal fragment is shown in two views in Figure 7. The surface in Figure 7a is highly charged, especially in the protruding loop regions 23 - 27, 35 - 40 and 64 - 66. The surface in Figure 7b contains more neutral hydrophilic residues as well as a small hydrophobic patch from Pro85 - Phe87 near the center of the surface. In the future, such information could assist in understanding how these different surfaces may be involved in interactions with the rest of the MSP-1 precursor, the processing proteinase, other proteins on the merozoite surface, or unknown targets on the erythrocyte or parasite vacuolar membrane surfaces. Primary sequence conservation The residues involved in the hydrophobic domain interface in P. falciparum are also shown in Figure 1, together with corresponding residues in MSP-1 of the less virulent human malaria parasite, P. vivax (Del Portillo et al, 1991 ; Gibson et al, 1992). Extensive conservation of the interface residues (with conservative substitutions) suggests that P. vivax and perhaps other Plasmodium species as well, may have a similar U-shaped EGF module pair arrangement. Another feature of the P. vivax sequence, also seen in other Plasmodium species, is the single disulphide bond deficiency in the first EGF-like domain resulting from the absence of cysteine residues equivalent to the P. falciparum Cysl2 and Cys28. P. falciparum dimorphic sites Five dimoφhic sites have been observed in the P. falciparum MSP-1 19 C-terminal fragment from different isolates (Qari et al, 1998). Several observations can be made about the position of these sites on the MSP-1 structure. Two sites, Glnl4/Glul4 and Lys61/Thr61, involve residues in relatively well-structured backbone regions, with surface-exposed hydrophilic or charged side-chains. A pair of adjacent sites, with the sequence variants Asn70 - Gly71/Ser70 - Arg71, occurs in the disordered loop of domain- 2, within a segment (residues 68 - 71) that has been shown to be highly mobile. The region from Glu65 to Lys73 also appears to be the most variable region among different Plasmodium species (Daly et al, 1992; Holder et al, 1992). Finally, the fifth site has a substitution between hydrophobic residues (Leu86/Phe86). This partially-exposed side- chain is located at the hydrophobic domain interface, and the conservative substitution is consistent with a role in this interaction. Example 2 - Mutation and Monoclonal Antibody Binding studies As a step towards understanding antibody interactions with the MSP-1 C-terminal fragment, the effect of engineered point mutations (within domain- 1) on antibody binding has been studied. Amino acid substitutions were made that consisted of radical changes. These radical changes consisted of, for example, replacing an aliphatic residue with a charged polar residue, replacing a positively charged side chain with a negatively charged side chain, replacing an amino acid with a large side chain with an amino acid with a smaller or no side chain (giycine). replacing a polar amino acid with a charged polar amino acid, replacing a polar amino acid with an aromatic amino acid, replacing a large aromatic amino acid with an amino acid with a small side chain, and replacing cysteine residues that are involved in disulphide bonds. Four individual amino acid substitutions shown in Figure 8, each completely abolish binding of one or more mAbs to the mutant fragment, as detected by Western blotting. The Glu26 mutation, shown in cyan, is closest to the N-terminal proteolytic processing site (magenta) at Asnl, and is the only one of this group of mutations that affects binding of a processing-inhibitory antibody, i.e. one that is capable of preventing both proteolytic processing of the MSP-1 precursor and erythrocyte invasion in vitro. The other three mutations abolish binding of blocking antibodies that bind to the native C-terminal fragment and interfere with the binding of processing-inhibitory antibodies. Additional mutations were made based on the immunochemical analyses and the tertiary structure of the molecule, and the binding of the mAbs was assessed by western blotting and BIAcore analysis. The results are summarised in Table 2. The results of the binding of selected mAbs to the modified proteins as detected by Western blotting are shown in Figure 9, and by BIAcore analysis in Figure 10. Some individual amino acid changes have no effect on the binding of any of the mAbs tested (for example Leu22 to Arg). Other substitutions affect the binding of one or more mAbs. Of particular interest are those changes that prevent the binding of blocking antibodies but have no effect on the binding of the inhibitory antibodies. For example, replacement of Asnl 5 by Arg prevents the binding of mAb 7.5, replacement of Glu27 by Tyr prevents the binding of mAb 2.2, replacement of Leu31 by Arg prevents the binding of mAb 1E1, replacement of Tyr34 by Ser prevents the binding of mAb 7.5, and replacement of Glu43 by Leu prevents the binding of mAb 11 1.4. Several combinations of substitutions that prevent the binding of blocking antibodies but do not affect the binding of inhibitory antibodies ' were made in single proteins (Table 2 and Figure 1 1). In the first Glu27→Tyr. Leu31→Arg and Glu43— >Leu were combined, in the second Glu27- Tyr. Leu31→Arg, Tyr34-»Ser, and Glu43— ->Leu were combined, and the third Asnl5→Arg, Glu27→Tyr, Leu31— Arg and Glu43→Leu were combined. None of these modified proteins bound any of the blocking antibodies but continued to bind the inhibitory antibodies. We propose that the mutant proteins will induce a polyclonal response that is more inhibitory than that induced by the wild type protein. The modified recombinant proteins will also be used to affinity select antibodies from pooled serum from individuals exposed to malaria. We hypothesise that the modified proteins will select less blocking antibody than the wild type protein and that therefore these selected antibodies will be more effective in inhibiting parasite invasion in vitro and secondary processing. In the first EGF-like domain of MSP-1 from the rodent, primate and P. vivax malaria parasites, cysteines 2 and 4 are not present. We have replaced this cysteine pair (Cys 12 and Cys28) in the P. falciparum protein. This does not have appear to have any effect on the binding of any of the inhibitory antibodies, but does abolish the binding of the blocking antibody mAb 2.2. We propose that one reason why the proteins from these other malaria parasites are more immunogenic is that T cell recognition is more effective or that processing by antigen processing cells proceeds by a different degradation pathway that drives the fine specificity of the antibody response in a more productive direction (see for example Egan et al, 1997). Removal of the cysteine pair may improve the immunogenicity of the modified protein and this will be assessed by measuring the level of antibodies induced by the P. falciparum protein without the two cysteines with the level of antibodies induced by the wild type protein. Table 2 - The location of amino acid sequence changes and their effect on the binding of monoclonal antibodies CΛ CΛ H H H W CΛ w H * ++ = strong binding, + = binding, - = no binding Table 2 (cont.) CΛ to CΛ H I— i H H W CΛ W H I ++ = strong binding, + = binding, - = no binding TABLE A =? 13-10-98 merozoite surface protein- 1 (MSP-1 ) Plasmodium falciparum (C-terminal fragment) ≠ Reference: Η : DSS=0.000 dioxane=3.755 (internal) =* ,5 N: indirect -4 I3 C: indirect n 25C pH 6.5 50mM NaP04 lOOmM NaCl 90%H2O/10%D2O # -FORMAT-- ≠ BioMagResBank u ≠ The original sequence entered was: u NISQHQCVKKQCPQNSGCFRHLDEREECKCLLNYKQEGDKCVENPNPTCNENNGGCDADAKCTEEDSGSNGK KITCECTKPDSYPLFDGIFCSSSN # # Expressed in NMR-STAR, this sequence is: _Mol_residue_sequence NISQHQCVKKQCPQNSGCFR H DEREECKCLLNYKQEGDK CVENPNPTCNENNGGCDADA KCTEEDSGSNGKKITCECTK PDSYPLFDGIFCSSSN loop_ _Residue_seq_code _Residue_author_seq_code Residue label 1 @ ASN 2 @ ILE 3 a SER 4 @ GLN 5 @ HIS 6 @ G N 7 @ CYS 8 3 VAL 9 @ LYS 10 @ LYS 11 @ GLN 12 @ CYS 13 @ PRO 14 @ GLN 15 @ ASN 16 @ SER 17 @ GLY 18 3 CYS 19 @ PHE 20 @ ARG 21 @ HIS 22 @ LEU 23 @ ASP 24 @ GLU 25 @ ARG 26 @ GLU 27 @ GLU 28 @ CYS 29 @ LYS 30 @ CYS 31 @ LEU 32 @ LEU 33 8 ASN 34 @ TYR 35 @ LYS 36 @ GLN 37 @ GLU 38 0 GLY 39 @ ASP 40 @ LYS 41 @ CYS 42 @ VAL 43 @ GLU 44 @ ASN 45 @ PRO 46 @ ASN 47 @ PRO 48 <? THR 49 @ CYS 50 @ ASN 51 @ GLU 52 @ ASN 53 @ ASN 54 @ GLY 55 @ GLY 56 @ CYS 57 @ ASP 58 @ ALA 59 @ ASP 60 @ ALA 61 @ LYS 62 @ CYS 63 @ THR 64 @ GLU 65 @ GLU 66 @ ASP 67 @ SER 68 @ GLY 69 @ SER 70 @ ASN 71 @ GLY 72 @ LYS 73 @ LYS 74 @ ILE 75 @ THR 76 @ CYS 77 @ GLU 78 @ CYS 79 @ THR 80 @ LYS 81 @ PRO 82 @ ASP 83 @ SER 84 @ TYR 85 @ PRO 36 @ LEU 87 @ PHE 88 @ ASP 89 @ GLY 90 @ ILE 91 @ PHE 92 @ CYS 93 @ SER 94 @ SER 95 @ SER 96 @ ASN stop_ *## ##### # Chemical Shift Ambiguity Code Definitions Codes Definition Unique Ambiguity of geminai atoms or geminai methyl proton groups .Aromatic atoms on opposite sides of the ring# (e.g. Tyr HE1 and HE2 protons) Intraresidue ambiguities (e.g. Lys HG and # HD protons) Interresidue ambiguities (Lys 12 vs. Lys 27) # Ambiguous, specific ambiguity not defined #################################################* INSTRUCTIONS # 1) Replace the @-sιgns with appropriate values. # 2) Text comments concerning the assignments can be supplied in the full deposition. # 3) Feel free to add or delete rows to the table as needed. # The row numbers (_Atom_shift_assign_ID values) will be re-assigned to sequential values by BMRB # # The atom table chosen for this sequence is: loop_ _Atom_shift_assign_ID _Residue_seq_code _Residue_label _Atom_name _Atom_type _Chem_shift_value _Chem_shift_value_error _Chem_shift_ambiguity_code # Table Al. Supplementary: IH, 13C and 15N chemical shift assignments of MSP-1 C-terminal fragment Atom Residue shift Seq Residue Atom Atom Shift/ Error/ Ambiguity- assign no. Name Name Type ppm ppm Code 1 ASN H H 8.29 0.02 1 2 ASN HA H 4.60 0.02 1 ASN HB2 H 2.86 0.02 2 4 ASN HB3 H 2.75 0.02 2 5 ASN HD21 H ? ? 6 ASN HD22 H 7 ? 7 ASN C C 7 7 9 8 ASN CA C 55.5 0.6 1 9 ASN CB C 40.9 0.6 1 1 1 ASN N N 125.8 0.3 1 12 ASN ND2 N 7 ? 7 13 2 ILE H H 8.29 0.02 1 2 ILE HA H 4.25 0.02 2 ILE HB H 1.97 0.02 -> ILE HG 12 H " 1.39 0.02 2 2- ILE HG13 H 1.19 0.02 -> ILE HG2 H 0.92 0.02 T ILE HD 1 H 0.81 0.02 7 ILE C C 173.8 0.6 -> ILE CA C 62.2 0.6 1 ILE CB C 38.7 0.6 2 ILE CGI C 27.5 0.6 2 ILE CG2 C 18.2 0.6 2 ILE CD1 C 13.7 0.6 2 ILE N N 121.1 0.3 3 SER H H 8.47 0.02 3 SER HA H 4.20 0.02 — . SER HB2 H 3.90 0.02 3 SER HB3 H 3.90 0.02 3 SER C C 7 ? 3 SER CA C 60.9 0.6 3 SER CB C 63.3 0.6 3 SER N N 119.3 0.3 4 GLN H H 8.32 0.02 4 GLN HA H 4.02 0.02 4 GLN HB2 H 1.88 0.02 GLN HB3 H 1.88 0.02 4 GLN HG2 H 1.75 0.02 GLN HG3 H 1.75 0.02 4 GLN HE21 H 7 ? 7 GLN . HE22 H ? ? 1 GLN C C ? 7 7 GLN CA C 57.7 0.6 1 GLN CB C 27.9 0.6 1 GLN CG C 32.7 0.6 1 GLN N N 121.6 0.3 1 GLN NE2 N ? ? HIS H H 7.76 0.02 9 HIS HA H 5.09 0.02 1 HIS HB2 H 2.70 0.02 1 HIS HB3 H 2.70 0.02 1 HIS HD2 H 6.87 0.02 1 HIS HE1 H 7.92 0.02 1 HIS C C 175.7 0.6 1 HIS CA C 54.8 0.6 1 HIS CB C 29.2 0.6 1 HIS N N 1 13.6 0.3 9 GLN H H 7.42 0.02 9 GLN HA H 4.43 0.02 1 GLN HB2 H 2.05 0.02 1 GLN HB3 H 2.05 0.02 1 GLN HG2 H 2.42 0.02 1 GLN HG3 H 2.42 0.02 1 GLN HE21 H 7.59 0.02 5 GLN HE22 H 6.92 0.02 5 GLN C C 175.7 0.6 1 GLN CA C 55.1 0.6 1 GLN CB C 28.8 0.6 1 GLN CG C 33.8 0.6 1 GLN N N 122.5 0.3 9 82 6 GLN NE2 N 1 12.6 0.3 5 83 7 CYS H H 9.18 0.02 1 84 7 CYS HA H " 4.09 0.02 1 85 7 CYS HB2 H 3.31 0.02 2 86 7 CYS HB3 H 3.1 1 0.02 2 88 7 CYS C C 174.4 0.6 1 89 7 CYS CA C 56.6 0.6 1 90 -7 CYS CB C 42.3 0.6 1 91 7 CYS N N 124.5 0.3 1 92 8 VAL H H 10.42 0.02 1 93 8 VAL HA H 4.33 0.02 1 94 8 VAL HB H 2.15 0.02 1 95 8 VAL HG 1 H 0.84 0.02 2 96 8 VAL HG2 H 0.82 0.02 2 97 8 VAL C C 176.6 0.6 1 98 8 VAL CA C 62.5 0.6 1 99 8 VAL CB C 34.4 0.6 1 100 8 VAL CG I C 21.5 0.6 2 101 8 VAL CG2 C 19.7 0.6 2 102 8 VAL N N 1 19.1 0.3 1 103 9 LYS H H 9.42 0.02 1 104 9 LYS HA H 4.51 0.02 1 105 9 LYS HB2 H 1.81 0.02 4 106 9 LYS HB3 H 1.81 0.02 4 107 9 LYS HG2 H 1.41 0.02 4 108 9 LYS HG3 H 1.41 0.02 4 109 9 LYS HD2 H 7 ? 9 1 10 9 LYS HD3 H 7 9 1 1 1 9 LYS HE2 H 3.33 0.02 1 1 12 9 LYS HE3 H 3.33 0.02 1 1 14 9 LYS C C ? 9 9 1 15 9 LYS CA C 57.7 0.6 1 1 16 9 LYS CB C ? 7 9 1 17 9 LYS CG C 9 0 7 1 18 9 LYS CD C 9 9 9 1 19 9 LYS CE C 9 9 9 120 9 LYS N N 124.1 0.03 1 122 10 LYS H H 8.94 0.02 1 123 10 LYS HA H 4.06 0.02 1 124 10 LYS HB2 H 1.86 0.02 1 125 10 LYS HB3 H 1.86 0.02 1 126 10 LYS HG2 H 1.29 0.02 1 127 10 LYS HG3 H 1.29 0.02 1 128 10 LYS HD2 H 1.70 0.02 2 129 10 LYS HD3 H 1.59 0.02 2 130 10 LYS HE2 H 3.04 0.02 1 131 10 LYS HE3 H 3.04 0.02 1 133 10 LYS C C ? 7 ? 134 10 LYS CA C 57.1 0.6 1 135 10 LYS CB C 34.3 0.6 1 136 10 LYS CG C 25.6 0.6 1 137 10 LYS CD C 29.6 0.6 1 138 10 LYS CE C 42.4 0.6 1 139 10 LYS N N 122.4 0.3 1 141 1 1 GLN H H 7 9 9 142 1 1 GLN HA H 4.47 0.02 1 143 1 1 GLN HB2 H 2.03 0.02 2 144 1 1 GLN HB3 H 1.89 0.02 2 o o o o o υι υι iΛ υι iΛ υι υι υι iΛ -ii 4. ■U 4-. 4 ■j ov iΛ <> j vo J. w — o vo oo -o σv on σv σv 4-. 4-. 4-. 4-. Oi Oi Oi Oi Oi Oi Oi i Oi Oi Oi Oi to to to to to to to to xxx^zonoxxxxxx^znnnnxxxxxxxxnnannxxxxxxxznnnxxxxz^nnnnxxxx on σv - to to 4-. bo 4-. io 4 4-. on OO OO o o o oo oo o o o o o o o o oo o o o o o o o o o o o o O o o • o • o o O o o o o to to to to Oi Oi Ov Ov Ov CTv O O O O O O O O Ov i Ov Ov OV O O O O O O O Ov o Oi σv σv σv o o o o to to to to to to t ) to to to to to to to to t to to t to to J- 208 16 SER HB3 H 3.52 0.02 210 16 SER C C 173.5 0.6 21 1 16 SER CA C 57.5 0.6 9 16 SER CB C 67.9 0.6 213 16 SER N N 109.9 0.3 214 17 GLY H H 8.92 0.02 215 17 GLY HA2 H 3.83 0.02 216 17 GLY HA3 H 2.06 0.02 217 17 GLY C C 7 9 218 17 GLY CA C 42.6 0.6 219 17 GLY N N 108.5 0.3 220 18 CYS H H 7.01 0.02 221 18 CYS HA H 5.63 0.02 222 18 CYS HB2 H 3.01 0.02 223 18 CYS HB3 H 3.01 0.02 225 18 CYS C C 172.5 0.6 226 18 CYS CA C 55.8 0.6 227 18 CYS CB C 43.2 0.6 228 18 CYS N N 120.5 0.3 229 19 PHE H H 9.12 0.02 30 19 PHE HA H 4.43 0.02 31 19 PHE HB2 H 1.70 0.02 9 32 19 PHE HB3 H 0.62 0.02 2 33 19 PHE HD 1 H 6.12 0.02 34 19 PHE HD2 H 6.12 0.02 35 19 PHE HE1 H 6.30 0.02 36 19 PHE HE2 H 6.30 0.02 37 19 PHE HZ H 6.37 0.02 38 19 PHE C C 172.3 0.6 39 19 PHE CA C 57.0 0.6 40 19 PHE CB C 41.7 0.6 47 19 PHE N N 132.0 0.6 48 20 ARG H H 7.76 0.02 49 20 ARG HA H 4.83 0.02 50 20 ARG HB2 H 1.26 0.02 2 51 20 ARG HB3 H 0.99 0.02 2 52 20 ARG HG2 H 1.59 0.02 2 53 20 ARG HG3 H 1.42 0.02 2 54 20 ARG HD2 H 3.35 0.02 2 55 20 ARG HD3 H 3.10 0.02 2 56 20 ARG HE H 7.18 0.02 1 57 20 ARG HH1 1 H 6.23 0.02 5 58 20 ARG HH12 H 6.23 0.02 5 59 20 ARG HH21 H 6.23 0.02 5 60 20 ARG HH22 H 6.23 0.02 5 61 20 ARG C C 174.7 0.6 1 62 20 ARG CA C 54.3 0.6 1 63 20 ARG CB C 32.3 0.6 1 64 20 ARG CG C 28.0 0.6 1 65 20 ARG CD C 44.1 0.6 1 67 20 ARG N N 129.4 0.3 1 68 20 ARG NE N 85.0 0.3 1 69 20 ARG NH1 N 70.1 0.3 5 70 20 ARG NH2 N 70.1 0.3 5 71 21 HIS H H 9.30 0.02 1 72 21 HIS HA H 4.50 0.02 1 73 21 HIS HB2 H 3.52 0.02 2 74 21 HIS HB3 H 3.44 0.02 2 to t to to to to t to to t to I io to to to to to to IO IJ t IJ IO to IJ to to to to to to to to to to to to to to to to to to to to IO IO tJ to to IO I to to to to IO to IO IO IO IO IO IO to o t J to to i io on on on on on on on on n on on on on On on on on on on 4 4- J 4-. 4-. 4- 4-. 4ι 4ι 4-. 4-. 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On On >— 405 30 CYS CB C 35.9 0.6 1 406 30 CYS N N 121.7 0.3 1 407 3 1 LEU H H " 7.81 0.02 1 408 31 LEU HA H 4.23 0.02 1 409 31 LEU HB2 H 1.39 0.02 1 410 31 LEU HB3 H 1.73 0.02 1 41 1 31 LEU HG H 0.94 0.02 1 412 31 LEU HD1 H 0.68 0.02 1 413 31 LEU HD2 H 0.77 0.02 1 414 31 LEU C C 176.2 0.6 1 415 31 LEU CA C 54.8 0.6 1 416 31 LEU CB C 42.5 0.6 1 417 31 LEU CG C 27.1 0.6 1 418 31 LEU CD1 C 26.0 0.6 1 419 31 LEU CD2 C 21.9 0.6 1 420 31 LEU N N 119.2 0.3 1 421 32 LEU H H 8.91 0.02 1 422 32 LEU HA H 4.26 0.02 1 23 32 LEU HB2 H 1.75 0.02 1 24 32 LEU HB3 H 1.32 0.02 1 25 32 LEU HG H 1.77 0.02 1 26 32 LEU HD1 H 0.96 0.02 1 27 32 LEU HD2 H 0.77 0.02 1 28 32 LEU C C 179.0 0.6 1 29 32 LEU CA C 56.4 0.6 1 30 32 LEU CB c 42.1 0.6 1 31 32 LEU CG c 26.9 0.6 1 32 32 LEU CD1 c 26.0 0.6 1 33 32 LEU CD2 c 23.4 0.6 1 34 32 LEU N N 118.4 0.3 1 35 ASN H H 8.84 0.02 1 36 33 ASN HA H 3.90 0.02 1 37 — . -. ASN HB2 H 3.26 0.02 1 38 33 ASN HB3 H 2.88 0.02 1 39 ASN HD21 H 6.86 0.02 1 40 33 ASN HD22 H 7.25 0.02 1 41 33 ASN C C 173.8 0.6 1 42 33 ASN CA C 56.9 0.6 1 43 33 ASN CB C 37.3 0.6 1 45 33 ASN N N 110.8 0.3 1 46 ASN ND2 N 112.0 0.3 1 47 34 TYR H H 8.61 0.02 1 48 34 TYR HA H 5.02 0.02 1 49 34 TYR HB2 H 3.35 0.02 1 50 34 TYR HB3 H 2.52 0.02 1 51 34 TYR HD1 H 6.74 0.02 1 52 34 TYR HD2 H 6.74 0.02 1 53 34 TYR HE1 H 6.69 0.02 1 54 34 TYR HE2 H 6.69 0.02 1 56 34 TYR C C 174.7 0.6 1 57 34 TYR CA C 57.6 0.6 1 58 34 TYR CB C 41.0 0.6 1 65 34 TYR N N 118.6 0.3 1 66 35 LYS H H 9.99 0.02 1 67 35 LYS HA H 4.84 0.02 1 68 35 LYS HB2 ~ H 1.82 0.02 1 69 35 LYS HB3 H 1.59 0.02 1 70 35 LYS HG2 H 1.18 0.02 1 471 35 LYS HG3 H 1.18 0.02 1 472 35 LYS HD2 H 1.48 0.02 1 473 35 LYS HD3 H 1.48 0.02 1 474 35 LYS HE2 H 2.89 0.02 1 475 35 LYS HE3 H 2.89 0.02 1 477 35 LYS C C 174.1 0.6 1 478 35 LYS CA C 54.2 0.6 1 479 35 LYS CB C 36.3 0.6 1 480 35 LYS CG C 24.1 0.6 1 481 35 LYS CD C 29.6 0.6 1 482 35 LYS CE C 41.6 0.6 1 483 35 LYS N N 1 19.7 0.3 1 485 36 GLN H H 8.77 0.02 1 486 36 GLN HA H 4.67 0.02 1 487 36 GLN HB2 H 2.06 0.02 1 488 36 GLN HB3 H 2.06 0.02 1 489 36 GLN HG2 H 2.35 0.02 1 490 36 GLN HG3 H 2.35 0.02 1 491 36 GLN HE21 H 7.50 0.02 2 492 36 GLN HE22 H 6.55 0.02 2 493 36 GLN C C 176.3 0.6 1 494 36 GLN CA C 56.7 0.6 1 495 36 GLN CB C 28.6 0.6 1 496 36 GLN CG C 33.7 0.6 1 498 36 GLN N N 124.9 0.3 1 499 36 GLN NE2 N 1 10.9 0.3 1 500 37 GLU H H 8.96 0.02 1 501 37 GLU HA H 4.51 0.02 1 502 37 GLU HB2 H 1.96 0.02 2 503 37 GLU HB3 H 1.80 0.02 2 504 37 GLU HG2 H 2.1 1 0.02 1 505 37 GLU HG3 H 2.1 1 0.02 1 506 37 GLU C C 176.2 0.6 1 507 37 GLU CA C 56.0 0.6 1 508 37 GLU CB C 31.9 0.6 1 509 37 GLU CG C 36.6 0.6 1 511 37 GLU N N 130.2 0.3 1 512 38 GLY H H 9.20 0.02 1 513 38 GLY HA2 H 3.69 0.02 1 514 38 GLY HA3 H 4.05 0.02 1 515 38 GLY C C 174.7 0.6 1 516 38 GLY CA C 47.4 0.6 1 517 38 GLY N N 118.4 0.3 1 518 39 ASP H H 8.81 0.02 1 519 39 ASP HA H 4.69 0.02 1 520 39 ASP HB2 H 2.78 0.02 1 521 39 ASP HB3 H 2.78 0.02 1 522 39 ASP C C 175.2 0.6 1 523 39 ASP CA C 54.2 0.6 1 524 39 ASP CB C 41.0 0.6 1 526 39 ASP N N 127.1 0.3 1 527 40 LYS H H 7.86 0.02 1 528 40 LYS HA H 4.71 0.02 1 529 40 LYS HB2 H 1.86 0.02 1 530 40 LYS HB3 H 1.86 0.02 1 531 40 LYS HG2 ' H 1.52 0.02 1 532 40 LYS HG3 H 1.52 0.02 1 533 40 LYS HD2 H 1.69 0.02 1 on on ιfl vO Oι υι 4- 4 4i 4ι 4. 4l 4i 4i 4i 4i 4i 4- 4l 4i 4». 4i 4i 4- 4- 4i .4- 4l 4i 4i 4- 4- 4-. 4-. 4-. 4i 4- 4-. 4- 4i 4l 4. 4-. 4. 4- 4- 4- 4- 4. 4- 4- 4- 4- 4- 4- 4- 4 4- 41 4 4 4 4 4ι 41 41 4 OJ OJ OJ OJ OJ OJ OJ Oi Oi Oi OJ t to to IO t IO ) to to to IO — — o o o o o o o o o o T) 73 , -0 , -0 , -0 -0 -0 > > > > > > > > > > ^ jα pa O JO JO pO ςn CΛ Λ CΛ CΛ Λ Λ >ooooooooαoo<<<<<<<<<<<o ooooooor , rr , r-r-r r'r- ooooooozzzzzzzzzz z cdcccccccc r r tΛ o x x x x X Z O O XXX zoooxxxxzooooooxxx Ooi Oto oroi Sto Oto O— CO > O O B ω> ω Oiω IO m o o ro > m Oi m tO oi χχχχχxx oooχχχχχχzoo oχχxχχχz;oooooχχχχχzoooχχχxzoonnooχ33X OJ ~ bo bo vo vo vo o σv — Ov -o - on to vo 0 0 0 0 0 0 0 0 0 0 0 -0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 O O O O O O O OJ OJ OV OV to to to to to to to to to to to to to lo io to to to to ιo to to to to t to t io lo to to •O IO to IO IO IO to IO to 597 45 PRO C C 176.2 0.6 598 45 PRO CA C 63.7 0.6 599 45 PRO CB 32.6 0.6 600 45 PRO CG C 26.8 0.6 601 45 PRO CD C 50.9 0.6 603 46 ASN H H 7.47 0.02 604 46 ASN HA H 5.09 0.02 605 46 ASN HB2 H 2.72 0.02 2 606 46 ASN HB3 H 2.43 0.02 2 607 46 ASN HD21 H 7.57 0.02 608 46 ASN HD22 H 6.91 0.02 609 46 ASN C C ? 7 610 46 ASN CA C 51.6 0.6 61 1 46 ASN CB C 40.1 0.6 613 46 ASN N N 1 14.0 0.3 614 46 ASN ND2 N 1 12.6 0.3 615 47 PRO HA H 4.34 0.02 616 47 PRO HB2 H 1.97 0.02 617 47 PRO HB3 H 1.77 0.02 618 47 PRO HG2 H 1.96 0.02 619 47 PRO HG3 H 1.96 0.02 620 47 PRO HD2 H 3.52 0.02 2 621 47 PRO HD3 H 3.39 0.02 2 622 47 PRO C C 175.5 0.6 623 47 PRO CA C 63.7 0.6 624 47 PRO CB C 32.4 0.6 625 47 PRO CG C 27.5 0.6 626 47 PRO CD C 49.8 0.6 628 48 THR H H 8.28 0.02 629 48 THR HA H 4.68 0.02 630 48 THR HB H 4.32 0.02 632 48 THR HG2 H 1.13 0.02 633 48 THR C C 174.5 0.6 634 48 THR CA C 59.1 0.6 635 48 THR CB C 69.9 0.6 636 48 THR CG2 C 19.3 0.6 637 48 THR N N 112.4 0.3 638 49 CYS H H 9.35 0.02 639 49 CYS HA H 4.44 0.02 640 49 CYS HB2 H 2.64 0.02 641 49 CYS HB3 H 3.14 0.02 643 49 CYS C C 175.8 0.6 644 49 CYS CA C 55.5 0.6 645 49 CYS CB C 37.7 0.6 646 49 CYS N N 125.3 0.3 647 50 ASN H H 8.36 0.02 648 50 ASN HA H 4.60 0.02 649 50 ASN HB2 H 2.88 0.02 650 50 ASN HB3 H 2.70 0.02 651 50 ASN HD21 H 7.60 0.02 2 652 50 ASN HD22 H 6.97 0.02 2 653 50 ASN C C 174.4 0.6 654 50 ASN CA C 54.8 0.6 655 50 ASN CB C 38.9 0.6 657 50 ASN N N 1 16.4 0.3 658 50 ASN ND2 N 1 13.6 0.3 659 51 GLU H H 7.43 0.02 660 51 GLU HA H 4.64 0.02 661 51 GLU HB2 H 1.96 0.02 2 662 51 GLU HB3 H 1.81 0.02 2 663 51 GLU HG2 H " 2.12 0.02 1 664 51 GLU HG3 H 2.12 0.02 1 665 51 GLU C C 176.1 0.6 1 666 51 GLU CA C 55.3 0.6 1 667 51 GLU CB C 31.5 0.6 1 668 51 GLU CG C 36.1 0.6 1 670 51 GLU N N 1 19.2 0.3 1 671 52 ASN H H 9.57 0.02 1 672 52 ASN HA H 4.45 0.02 1 673 52 ASN HB2 H 3.14 0.02 1 674 52 ASN HB3 H 2.64 0.02 1 675 52 ASN HD21 H 6.99 0.02 1 676 52 ASN HD22 H 7.71 0.02 1 677 52 ASN C C 176.4 0.6 1 678 52 ASN CA C 54.2 0.6 1 679 52 ASN CB C 37.8 0.6 1 681 52 ASN N N 125.4 0.3 1 682 52 ASN ND2 N 112.8 0.3 1 683 53 ASN H H 9.31 0.02 1 684 53 ASN HA H 4.64 0.02 1 685 53 ASN HB2 H 3.25 0.02 1 686 53 ASN HB3 H 2.31 0.02 1 687 53 ASN HD21 H 6.95 0.02 1 688 53 ASN HD22 H 6.26 0.02 1 689 53 ASN C C 176.2 0.6 1 690 53 ASN CA C 54.1 0.6 1 691 53 ASN CB C 39.2 0.6 1 693 53 ASN N N 119.3 0.3 1 694 53 ASN ND2 N 111.6 0.3 1 695 54 GLY H H 7.92 0.02 1 696 54 GLY HA2 H 4.28 0.02 1 697 54 GLY HA3 H 3.67 0.02 1 698 54 GLY C C 173.0 0.6 1 699 54 GLY CA C 46.3 0.6 1 700 54 GLY N N 106.3 0.3 1 701 55 GLY H H 8.06 0.02 1 702 55 GLY HA2 H 4.39 0.02 1 703 55 GLY HA3 H 3.47 0.02 1 704 55 GLY C C 175.1 0.6 1 705 55 GLY CA C 44.4 0.6 1 706 55 GLY N N 105.3 0.3 1 707 56 CYS H H 7.77 0.02 1 708 56 CYS HA H 4.39 0.02 1 709 56 CYS HB2 H 3.12 0.02 1 710 56 CYS HB3 H 2.81 0.02 1 712 56 CYS C C 175.2 0.6 1 713 56 CYS CA C 53.1 0.6 1 714 56 CYS CB C 36.6 0.6 1 715 56 CYS N N 117.6 0.3 1 716 57 ASP H H 8.37 0.02 1 717 57 ASP HA H 3.98 0.02 1 718 57 ASP HB2 H 2.24 0.02 2 719 57 ASP HB3 . H 2.03 0.02 2 720 57 ASP C C 176.0 0.6 1 721 57 ASP CA C 56.0 0.6 1 722 57 ASP CB C 45.0 0.6 1 724 57 ASP N N 122.3 0.3 725 58 ALA H H 8.48 0.02 726 58 ALA HA H 3.98 0.02 727 58 ALA HB H 1.41 0.02 728 58 ALA C C 178.9 0.6 729 58 ALA CA C 55.6 0.6 730 58 ALA CB C 19.1 0.6 731 58 ALA N N 127.2 0.3 732 59 ASP H H 9.1 1 0.02 733 59 ASP HA H 4.90 0.02 734 59 ASP HB2 H 2.44 0.02 735 59 ASP HB3 H 2.82 0.02 736 59 ASP C C 174.6 0.6 737 59 ASP CA C 53.9 0.6 738 59 ASP CB C 40.7 0.6 740 59 ASP N N 1 16.7 0.3 741 60 ALA H H 7.94 0.02 742 60 ALA HA H 5.13 0.02 743 60 ALA HB H 1.27 0.02 744 60 ALA C C 176.3 0.6 745 60 ALA CA C 50.5 0.6 746 60 ALA CB C 21.3 0.6 747 60 ALA N N 121.9 0.3 748 61 LYS H H 9.06 0.02 749 61 LYS HA H 4.54 0.02 750 61 LYS HB2 H 1.77 0.02 751 61 LYS HB3 H 1.77 0.02 752 61 LYS HG2 H 1.31 0.02 2 753 61 LYS HG3 H 1.39 0.02 2 754 61 LYS HD2 H 1.72 0.02 2 755 61 LYS HD3 H 1.63 0.02 2 756 61 LYS HE2 H 2.92 0.02 2 757 61 LYS HE3 H 2.92 0.02 2 759 61 LYS C C 175.7 0.6 760 61 LYS CA C 55.1 0.6 761 61 LYS CB C 34.1 0.6 762 61 LYS CG C 24.6 0.6 763 61 LYS CD C 29.1 0.6 764 61 LYS CE C 42.1 0.6 765 61 LYS N N 122.5 0.3 767 62 CYS H H 9.19 0.02 768 62 CYS HA H 5.32 0.02 769 62 CYS HB2 H 2.44 0.02 770 62 CYS HB3 H 2.80 0.02 772 62 CYS C C 174.5 0.6 773 62 CYS CA C 56.1 0.6 774 62 CYS CB C 37.6 0.6 775 62 CYS N N 131.5 0.3 776 63 THR H H 9.23 0.02 777 63 THR HA H 4.51 0.02 778 63 THR HB H 4.01 0.02 780 63 THR HG2 H 1.16 0.02 781 63 THR C C 172.2 0.6 782 63 THR CA C 62.8 0.6 783 63 THR CB _ C 71.5 0.6 784 63 THR CG2 C 22.0 0.6 785 63 THR N N 125.9 0.3 786 64 GLU H H 8.61 0.02 u. lΛ υι U Uι Uι Uι 4. 4l 4. . . fc -(. 4. 4. 4. O O O O O O O O O Q O O O C C CC C dCC C C CC X Z o o o X 20 X Z o o o o p o x x Z o o o o x *- -- X Z O O O X X X X ω > X X x z • J ω > o ω > ro > X X X JU (- ω ro o ro > O O CO > O o ω co to OJ o to ro OJ ω IO > σv oxxxxxxzoooxxxxzooxxxzoooxxxxzoooxxxxzooooxxxxxxzooooxxxxx IO o io oi o/i lo to •— .— on si σv to 4 Oi Oi vo vo •— ' O oo o o o O IO o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o oi σv σv σv o o o o oi o o o oi σv σv Λ o o o i σv σv σv oo oo oo oo oi ov σv σv o o o o o o oi σv σv o o o o o to to to to to t to to to to t to to to to to to to to t to to to to to to i to to IO IO IO - - V O vO V O V O VO V O „ _- — - o o o I - O O M sl sl sl sl sl sj sj sl sl sj sl s] sl sl sl sl s| sl sl sl sl sl s| sj sl sl sl sl sl sl sl sl sl sl sl sl sl sl sl sl sl s-| sl sl sl s| sl sl sϊ si si si sj si si i si si si 4 41 4 41 4 4- 4- 4- 41 41 41 4ι 41 4ι OJ OJ OJ OJ OJ OJ OJ OJ OJ OJ OJ OJ OJ Oi i OJ OJ to to to to to to IO to IO to to to to to IO 10 to 0 0 0 μ μμμμμμ μμμμμμμr < r rr" r rr , r z o o o o o o X X X X xzoooooo Z O O O O O O Z X X Z -Z O o o o ro > D m α O CO > — O to •— Ω — m Oim tOσ Oio too OJα toro OJro to - tn σ o ω > s - s X t -o O _J o to oJ o to ro ro to > > α ro zonnonoxxxxxxxzooooooxxxxxxxxxxzoonoooxxxxxxxxxxzonxpπx z.00 ►— ■— to on -- 1— 1 .— • — ' 1— 41 00 — - to oj on i— to to — ►— — ■— ,— .-. 41 00 1— 4ι to ιo o on — to to .— — •— — — — 41 oo — 41 - 41 4- o 1— i υi O vo si 00 00 si on o 41 on on si bo o v v to ό j si si cΛ bo y ∞ .-^ ^ ^ ^ vo vo σΛ σ -i lΛ si bo ό j g ' ^ o — 00 Oi si o — 41 σv 41 o vo vo OJ OJ On Ov — ' .— 41 — . I vo vo 0 4i . OJ OJ Ov Ov OO Ov vo on 4l vo . 0 . s-l sl CO si .— si O O O O O O O OO O O O O O O o - OO O O O O O OOO O O O O O O O O O O O O O O O OO O O O O OO O O O O 0 0 0 oi σv σv σv ov σv σv o o o o o o o oi σv σv σv σv o o o o o o o o o i σv σv σv σv σv σv o o o o o o o o o o oi αv σv o o o όi σv σv to t t t to to to to to to t to to to to to to to to N) to to to to to to to to to to t 10 10 10 913 75 THR H H 8.88 0.02 914 75 THR HA H 4.51 0.02 915 75 THR HB H " 4.01 0.02 917 75 THR HG2 H 1.15 0.02 918 75 THR C C 172.4 0.6 919 75 THR CA C 61.2 0.6 920 75 THR CB C 71.5 0.6 921 75 THR CG2 C 22.0 0.6 922 75 THR N N 120.3 0.3 923 76 CYS H H 8.82 0.02 924 76 CYS HA H 5.53 0.02 925 76 CYS HB2 H 3.28 0.02 926 76 CYS HB3 H 2.69 0.02 928 76 CYS C C 174.7 0.6 929 76 CYS CA C 52.2 0.6 930 76 CYS CB C 40.6 0.6 931 76 CYS N N 121.5 0.3 932 77 GLU H H 8.64 0.02 933 77 GLU HA H 4.74 0.02 934 77 GLU HB2 H 1.94 0.02 935 77 GLU HB3 H 1.94 0.02 936 77 GLU HG2 H 1.83 0.02 937 77 GLU HG3 H 1.83 0.02 938 77 GLU C C 175.7 0.6 939 77 GLU CA C 54.7 0.6 940 77 GLU CB C 33.1 0.6 941 77 GLU CG C 36.1 0.6 943 77 GLU N N 123.0 0.3 944 78 CYS H H 9.71 0.02 945 78 CYS HA H 4.48 0.02 946 78 CYS HB2 H 2.63 0.02 947 78 CYS HB3 H 3.30 0.02 949 78 CYS C C 175.8 0.6 950 78 CYS CA C 57.4 0.6 951 78 CYS CB C 39.2 0.6 952 78 CYS N N 129.1 0.3 953 79 THR H H 8.20 0.02 954 79 THR HA H 4.25 0.02 955 79 THR HB H 4.25 0.02 957 79 THR HG2 H 1.25 0.02 958 79 THR C C 176.2 0.6 959 79 THR CA C 63.4 0.6 960 79 THR CB C 70.1 0.6 961 79 THR CG2 C 22.2 0.6 962 79 THR N N 1 14.7 0.3 963 80 LYS H H 8.56 0.02 964 80 LYS HA H 4.50 0.02 965 80 LYS HB2 H 1.79 0.02 966 80 LYS HB3 H 1.79 0.02 967 80 LYS HG2 H 1.47 0.02 9 968 80 LYS HG3 H 1.64 0.02 9 969 80 LYS HD2 H 1.70 0.02 970 80 LYS HD3 H 1.70 0.02 971 80 LYS HE2 H 2.94 0.02 972 80 LYS HE3 H 2.94 0.02 974 80 LYS C C 7 9 975 80 LYS CA C 55.4 0.6 1 976 80 LYS CB C 30.9 0.6 i i — . o o vo vo vo vo vo v o o vo v vo o o o o o o o o o o o o o o o o o o o o o O vo OO OO OO OO OO OO Oo OO OO sl sl sl „ O vo oo si σs on 4 OJ IO O vO OO sl si σv σv oil il il on 0 Uι 0n o i ι θn 0n n 4 4 4 i 4ι 4 4ι 4ι 4 4 i 4ι 4 4ι 0J OJ O J 0J 0J 0 J tO tO t tO tO 0 t tO ι— -- — — ■ — — ^- — ^- "— — ^— O O O O PC X z χχ χooooo ρc x xx x x x Z O OJ t ωo ooco> oσooωro > m α o x xnonooxxx x x x xzoooxxx x xxxxzooox xx xzonπxxxxoononxx xxx xx zono 4 oo on to OJ σv — OJ OJ to to — to on —• OJ on σv σv σv σv to to on oo ■— σv on — OJ OJ J>. — 41 o;ι o to lo 4 oo on lo OJ σv OJ OJ IO to — IO 4- — 4ι IO IO O -si to to si OO OO IO tJ si to o to vo 4i σv σv vo vo si vo o si fr-j y y Hr-* ^rj oonn oonn 4i — O on si OO to si O si — 4 si σv vo o P- - bo j io to to vo on si — σv OJ oo vo on on lo to to on vo W si σv vo vo to to on OJ — to ϊ^ VO 4I . O on si si oo on oo oo oo vo to . oj O 4 θ s-i si ov r 41" — to vo 41 P- O O O O O O O O O O O O O O O O O o o o o o 0 0 0 0 0 0 0 0 o o o o o o o o o o p o o p o o o o o o o o o o o o o v bv ov bv v o o o o o o o o ov bv o o o o o o o o OJ σv σv σv o o o o OJ σv σv o o o o bv v ov bv bv o o o o o o o oj ov v bv to to IO IJ to io to to to to t to IO io to to to to to to to to to to to to to to to IO to to io to — — 1047 86 LEU HB2 H 1.76 0.02 1048 86 LEU HB3 H 1.76 0.02 1049 86 LEU HG H 1.85 0.02 1050 86 LEU HD 1 H 1.00 0.02 1051 86 LEU HD2 H 1.00 0.02 1052 86 LEU C C 177.9 0.6 1053 86 LEU CA C 54.5 0.6 1054 86 LEU CB C 44.8 0.6 1055 86 LEU CG C 29.0 0.6 1056 86 LEU CD 1 C 25.7 0.6 1057 86 LEU CD2 C 25.7 0.6 1058 86 LEU N N 120.2 0.3 1059 87 PHE H H 9.29 0.02 1060 87 PHE HA H 4.16 0.02 1061 87 PHE HB2 H 3.26 0.02 1062 87 PHE HB3 H 3.19 0.02 1063 87 PHE HD1 H 7.31 0.02 1064 87 PHE HD2 H 7.31 0.02 1065 87 PHE HE 1 H 7.70 0.02 1066 87 PHE HE2 H 7.70 0.02 1067 87 PHE HZ H 7.67 0.02 1068 87 PHE C C 176.4 0.6 1069 87 PHE CA C 59.3 0.6 1070 87 PHE CB C 36.9 0.6 1077 87 PHE N N 122.6 0.3 1078 88 ASP H H 8.93 0.02 1079 88 ASP HA H 4.33 0.02 1080 88 ASP HB2 H 3.1 1 0.02 1081 88 ASP HB3 H 3.02 0.02 1082 88 ASP C C 175.2 0.6 1083 88 ASP CA C 56.3 0.6 1084 88 ASP CB C 39.9 0.6 1086 88 ASP - N N 1 1 1.3 0.3 1087 89 GLY H H 7.87 0.02 1088 89 GLY HA2 H 3.48 0.02 1089 89 GLY HA3 H 4.08 0.02 1090 89 GLY C C 174.7 0.6 1091 89 GLY CA C 46.0 0.6 1092 89 GLY N N 102.0 0.3 1093 90 ILE H H 7.17 0.02 1094 90 ILE HA H 4.39 0.02 1095 90 ILE HB H 1.52 0.02 1096 90 ILE HG12 H 0.65 0.02 2 1097 90 ILE HG13 H 0.65 0.02 2 1098 90 ILE HG2 H 1.05 0.02 1099 90 ILE HD1 H 0.51 0.02 1 100 90 ILE C C 175.1 0.6 1 101 90 ILE CA C 64.2 0.6 1 102 90 ILE CB C 35.9 0.6 1 103 90 ILE CG I c 25.5 0.6 1 104 90 ILE CG2 c 16.9 0.6 1 105 90 ILE CD 1 c 14.9 0.6 1 106 90 ILE N N 1 13.3 0.3 1 107 91 PHE H H 7.43 0.02 1 108 91 PHE HA H 5.15 0.02 1 109 91 PHE HB2 H 2.40 0.02 1 1 10 91 PHE HB3 H 2.40 0.02 1 1 1 1 91 PHE HD 1 H 7.00 0.02 1112 91 PHE HD2 H 700 002 1113 91 PHE HE1 H 695 002 1114 91 PHE HE2 FT 695 002 1115 91 PHE HZ H 706 002 1116 91 PHE C C 1751 0.6 1117 91 PHE CA C 565 06 1118 91 PHE CB C 443 06 1125 91 PHE N N 1140 03 1126 92 CYS H H 786 002 1127 92 CYS HA H 5.18 0.02 1128 92 CYS HB2 H 2.57 0.02 1129 92 CYS HB3 H 3.08 0.02 1131 92 CYS C C 1741 06 1132 92 CYS CA C 54.1 0.6 1133 92 CYS CB C 44.7 0.6 1134 92 CYS N N 119.0 0.3 1135 93 SER H H 9.26 0.02 1136 93 SER HA H 4.23 0.02 1137 93 SER HB2 H 3.90 0.02 1138 93 SER HB3 H 3.90 0.02 1140 93 SER C C 9 9 1141 93 SER CA C 60.4 0.6 1142 93 SER CB C 63.7 0.6 1143 93 SER N N 118.3 0.3 1144 94 SER H H 8.04 0.02 1145 94 SER HA H 4.59 0.02 1146 94 SER HB2 H 3.91 0.02 1147 94 SER HB3 H 3.91 0.02 1149 94 SER C C 7 9 9 1150 94 SER CA C 58.7 0.6 1151 94 SER CB C 64.0 0.6 1152 94 SER N N 114.2 0.3 1153 95 SER H H 9 9 9 1154 95 SER HA H 471 0.02 1 1155 95 SER HB2 H 4.02 0.02 2 1156 95 SER HB3 H 3.94 0.02 2 1158 95 SER C C ? 9 9 1159 95 SER CA C 58.4 0.6 1 1160 95 SER CB C 64.5 0.6 1 1161 95 SER N N 9 7 7 1162 96 ASN H H ? 7 9 1163 96 ASN HA H 4.61 0.02 1 1164 96 ASN HB2 H 2.75 0.02 2 1165 96 ASN HB3 H 2.60 0.02 2 1166 96 ASN HD21 H 9 9 9 1167 96 ASN HD22 H 9 7 9 1168 96 ASN C C 9 9 9 1169 96 ASN CA C 54.3 0.6 1 1170 96 ASN CB C 418 0.6 1 1172 96 ASN N N 9 9 9 1173 96 ASN ND2 N 9 9 9 stop_ - The following loop is used to define sets of Atom-shift assignment IDs that represent related ambiguous assignments taken from the above list of assigned chemical shifts. Each element in the set should be separated by a comma, as shown in the example below, and is the assignment ID for a chemical shift assignment that has been given as ambiguity code of 4 or 5 Each set indicates that the observed chemical shifts are related to the defined atoms, but have not been assigned uniquely to a specific atom in the set looρ_ Atom shift assisn ID ambisuitv - Sets of Atom-shift Assignment Ambiguities 4 ≠ Example- 5,4,7 u @ stop_ — REMARKS — =? PROTECTED BACKBONE AMIDE GROUPS - (SLOWLY EXCHANGING IN D20) FOR RESIDUES = GLY 17, PHE 19, GLU 27, LYS 29, LEU 31 , =? TYR 34 , LYS 35 , VAL 42 , CYS 56 , ASP 57 , - ALA 60, LYS 61 , THR 63, THR 75, GLU 77, =f LEU 86, GLY 89, ILE 90, PHE 91 # BROAD HN SIGNALS IN [15-Nj-HSQC OBSERVED FOR RESIDUES. ≠ VAL 8, LYS 9, LYS 10, CYS 18, ARG 20 # TWO BACKBONE HN CROSSPEAKS OBSERVED FOR RESIDUES. # HIS 5.778,113.8/ 774,113.5 ϋ GLN 6 -744,122.6/740,122.4 =? TWO AVERAGED NHVHH* SIGNALS OBSERVED FOR RESIDUES # ARG 20, ARG 25 NOT SPECIFICALLY ASSIGNED TO INDIVIDUAL ARGININES ή LYS 29 NZ/HZ* SIGNAL: TENTATIVELY ASSIGNED TO # LYS 29 (BURIED LYSINE SIDE CHAIN) # BASED ON GREATER PROTECTION FROM H20 EXCHANGE ≠ THAN OTHER LYSINE NZ/HZ* SIGNALS # ASPARAGINE SIDE CHAIN AMIDE SIGNALS. # PROBABLE OVERLAPPING CROSSPEAKS - 112 PPM [ 15 N] ≠ FOR ASN 1 , ASN 70 , ASN 96 ########## ######## s t####### ################## ########ff### ################# S !# ff # ^#######^##### # ####################^####### ########**^####^ #^# ###### ? i##### Table A2. Supplementary: NMR experimental details Experiment Dimension Nucleus Complex Spectral Acquisition Carrier Instrument Solvent Temp- Final Digital Mixing Total Points width Time Frequency Η- frequency erature data size Resolution time time [after LP] (points) (Hz) (ms) (ppm) (MHz) ro (points) (Hz/ (ms) (hr point) 2D NOESY tl Ή 400 8000 50 4.74 600 D,0 25 1024 7.8 75- 150 22 t2 Ή 2048 8000 256 4.74 2048 3.9 2D NOESY t l Ή 360 8000 45 4.74 600 H,0 25 1024 7.8 75 23 t2 Ή 2048 8000 256 4.74 2048 3.9 2D ROESY t l Ή 260 6000 43 4.74 500 D 2 0 25 1024 5.9 60 16 : t2 Ή 2048 6000 341 4.74 2048 2.9 2D ROESY t l Ή 360 7000 54 4.74 500 H,0 25 1024 6.8 60 62 t2 Ή 2048 7000 293 4.74 2048 3.4 3D [ 15 N]-NOESY-HSQ t l ,5 N 36 [64] 2500 14.4 121.5 500 H 2 0 25 128 19.5 125 64 t2 Ή 180 7000 26 4.74 512 13.7 t3 Ή 512 7000 73 4.74 5 12 13.7 3D [' 5 N]-R0ESY-HSQC tl ,5 N 32 2500 12.8 121.5 500 H-,0 25 128 19.5 60 87 t2 Ή 180 7000 26 4.74 512 13.7 t3 Ή 512 7000 73 4.74 512 13.7 3D [ n C]-I IMQC-NOESY t l Ή 160 7200 22 4.74 600 D 2 0 25 512 14.1 125 89 t2 ,3 C 96 10000 9.6 41.0 256 39.1 t3 Ή 384 7200 53 4.74 1024 7.0 4D [ 13C]-I IMQC-NOL SY-[ 13C]-I ISQC t l l3 C 18 [24] 3360 5.4 40.2 600 D,0 25 64 52.5 125 10: t2 Ή 74 3600 21 3.00 256 14.1 t3 l3 C 18 [24] 3360 5.4 40.2 64 52.5 t4 Ή 256 4500 57 3.00 256 17.6 Experiment Dimension Nucleus Complex Spectral Acquisition Carrier Instrument Solvent TemperFinal Digital Mixing Total {Reference} Points width Time Frequency Η-frequency ature data size Res. time time [after LP] (points) (Hz) (ms) (ppm) (MHz) (oC) (points) (I lz/ po int)(ms) (hr) 4D [ ,3 C]-HMQC- NOESY [ l5 N]-HSQC tl ,3 C 14 [24] 3360 4.2 40.2 600 H 2 0 25 64 52.5 125 96 t2 Ή 56 3600 15.6 3.00 128 28.1 t3 ,5 N 14 [24] 1800 7.8 1 18.9 64 28.1 Λ t4 'I ! 256 7400 34.6 4.74 256 29.0 td 2D DQF-COSY CΛ tl Ή 1000 6000 167 4.74 500 D 2 0 25 4096 1.5 31 H h- 1 t2 Ή 2048 6000 341 4.74 8192 0.7 H 3D HNI IA H tl "N 35 2500 14 121.5 500 H 2 0 25 128 19.5 41 W Λ t2 Ή 80 3500 23 4.74 256 13.7 13 Ή 512 7000 73 4.74 512 13.7 M 3D HNHB H tl "N 24 [48] 2500 9.6 1 19.1 600 H 2 0 25 128 19.5 60 i , t2 Ή 90 8000 1 1.3 4.74 256 31.3 -— • 1 t3 Ή 512 8000 64 4.74 1024 7.8 3D HN(CO)HB > tl l5 N 28 [48] 1800 15.6 1 18.9 600 H 2 0 25 128 14.1 108 ON t2 Ή 128 8000 16 4.74 512 15.6 t3 Ή 512 8000 64 4.74 512 15.6 2D [ l5 N]-[ 13 ' Cγ] Spin- ■ echo HSQC tl ,5 N 80 1800 44.4 1 18.9 600 H 2 0 25 256 7.0 13 t2 Ή 1312 8000 149 4.74 2048 3.9 2D [ l3 C'H 13 Cγ] Spin -echo HSQC tl ,5 N 78 1800 43.3 1 18.9 600 H 2 0 25 256 7.0 26 t2 Ή 1216 8000 152 4.74 2048 3.9 3D LRCH tl ,3 C 34 3017 1 1.3 17.9 600 D 2 0 25 256 1 1.8 84 t2 Ή 57 4800 1 1.9 2.25 256 18.8 (3 Ή 384 4000 96 2.25 1024 3.9 Experiment Dimension Nucleus Complex Spectral Acquisition Carrier Instrument Solvent Temper- Final Digital Mixing Total {Reference} Points width Time Frequency Η-frequency ature data size Resolution time time [after LP] (points) (Hz) (ms) (ppm) (MHz) (°C) (points) (Hz/ point) (ms) (hr) 2D TOCSY tl Ή 360 8000 45 4.74 600 H,0 25 1024 7.8 66 t2 Ή 2048 8000 256 4.74 2048 3.9 2D [ ,5 N]-HSQC tl ,5 N 360 4400 82 1 19.6 600 H 2 0 25 2048 2.1 14 t2 Ή 1216 8000 152 4.74 4096 2.0 2D [ 13 C]-HSQC tl ,3 C 400 12000 33.3 41.3 600 H 2 0 25 1024 1 1.7 2.7 t2 Ή 1216 8000 152 4.74 4096 2.0 3D [ l5 N]-TOCSY-HSQ tl l5 N 38 [64] 2500 15.2 1 19.0 600 H 2 0 25 128 19.5 56 43 a Ή 180 8000 22.5 4.74 512 15.6 t3 Ή 512 8000 64 4.74 512 15.6 3D HCCH-TOCSY tl Ή 134 5500 24.4 4.74 500 D 2 0 25 512 10.7 17 65 t2 ,3 C 128 8049 15.9 41.9 512 15.7 t3 Ή 416 5500 76 4.74 512 10.7 3D CBCA(CO)NH tl ,3 C 38[64] 10000 3.8 41.3 600 H 2 0 25 128 78.1 21 t2 "N 26[36] 1800 14.4 1 18.9 128 14.1 t3 Ή 512 8000 64 4.74 512 15.6 3D CBCANH tl l3 C 63[128] 10000 6.3 41.3 600 H 2 0 25 512 19.5 t2 ,5 N 26[52] 1800 14.4 1 18.9 128 14.1 t3 Ή 512 8000 64 4.74 256 31.3 TABLE B merozoite s rrace :ar_:m C-teminal fraσπ". X-PLOR format 09- 11- 98 noes - roes -- acr-rcximate- 5 0 tot l .ong-range 135 -eαium-range sequential 222 mrrares cue ~3 nyαrogen conαs 10 ι 2Q restraints/ pseudoatom corrections not used -- for R-6 averaging/summation AVERAGING: 1 class rtδs 5UM 1 class nsam SUM 1 class sing 1 class hbnd 1 types d methylene 1 <res αue-atom 1> <resιdue- atom 2> <dιst -mmus-plus> <type> class rt6s assign resid ^ and name nb#) (resid 19 and name hd#) 3 .6 3 6 0 0 aro ;_ assign resid 5 ana name riDff ) resid 19 ana name he ) 6 3 6 0 0 arom " 1 assign resid 1 Ξ and name ha) (resid 34 and name har) 3 6 3 6 0 0 arom ... assign resiα 1 =, ana name na) resiα 34 and name he?) 3 6 3 6 0 0 arom _ assign resid 17 ana name na=f ) resiα 37 ana name has ) 5 5 5 5 0 0 arom _ assign resiα 17 and name ha* ) resid 87 and name he#) 5 5 5 5 0 0 arom ~ 1 assign resid 19 and name hd#) resid 20 and name hn) 5 5 5 5 0 0 arom s assign resid 19 and name hd#) resid 21 and name hn) 5 5 5 5 0 0 arom _s assign resiα 19 and name hd#) resid 21 and name hd2) 3 6 3 6 0 0 arom m assign resid 19 and name hd#) resid 22 and name hd#) 3 6 3 6 0 0 arom n assign resid 19 and name hα#) resiα 86 and name ha) 5 5 5 5 0 0 a-rom assign resid 19 ana name hd#) resid 91 and name hb#) 3 6 3 6 0 0 arom __ assign resid 19 ana name hd#) resid 91 and name hd#) 3 6 3 6 0 0 arom _ assign resid 19 and name hd#) resid 91 an name he#) 5 3 5 5 0 0 arom __ assign resid 19 and name he#) resid 21 ana name hd2) 5 5 5 5 0 0 arom m assign resid 19 and name ne#) resid 22 ana name hd#) 3 6 3 6 0 0 arom ~ n assign resid 19 ana name he#) resid 86 and name hg) 5 5 5 0 0 arom " l assign resiα 19 and name ne=; resiα 91 and name ho#) "5 6 3 6 0 0 arom _ assign resid 19 and name he=f ) resid 91 ana name hd#) 5 5 5 5 0 0 arom __ assign resid 31 and name hn; resiα 34 and name hd#. 3 6 3 6 0 0 arom " r assign resiα 31 ana name ng) res d 34 ana name ndtf ) c 5 c 5 0 0 arom T assign resiα Ξ and name r.αi ) resid 3- ana name nd?) ε 5 5 5 0 0 arom " ~ assign resiα 2 - and name hα ϋ resiα 24 and name he* ) 5 -. - 5 0 0 arom " - assign resiα 31 and name nd2#) resid 37 ana name hdff) -> 8 2 8 0 0 arom ..-_ assign resiα 32 ana name hnj resiα 34 ana name hd#) 5 5 5 5 0 Λ arom ^ assign resiα 34 ana name nn) resid 34 ana name nd») 3 6 3 6 0 0 arom i. assign resiα 34 and name na i resiα 34 ana name hdif) 3 3 6 0 0 arom _ assign ( resid 34 ana name hα= resiα 35 and name n ) Ξ 6 3 6 0 0 arom 5 assign resid - ana name πα* ) 'resiα ^2 ana name nni 5 5.5 n .0 ' arom 1 assign resiα Ξ - and name ια#) (resiα ^2 ana name na j 3 .6 3 .6 arom 1 assign resiα - and name r.e*) ( resiα - 5 ana name nn) 5 .5 5 .5 0 ' arom _ assign resiα 5 - and name nα# ' resiα _ 3 a a name no?; 5 .5 5 .5 0 arom _ assign resid 3ι ana name neif ) 1 resiα 42 ana name nn) z .5 5 0 c arom ± assign resiα and name -leϊ) resiα ^ 2 and name na) 5 .5 5 .3 c .0 1 rom 1 ass gn resiα and name ne*) resiα •» 3 ana name ho*) 3 .5 5 .5 0 .0 1 arom 1 ass_gn 'resiα Z - and name r.e* 1 , res_α 43 and name hg* i 3 .6 3 .6 0 .0 1 rom i assign ' resiα 33 and name nn \ ' resiα 34 ana name hds) 3 .6 3 .6 0 1 arcm s assign iresiα 36 ana name nn) resiα 51 and name hd#) 2 .5 5 .5 0 .0 arom 1 assign ' resid 00 and name nc ) ( resid 37 and name hα#) 2 .8 2 .8 0 .0 arom s assign 'resiα 36 and name hD#) ι ~ s iα 3 " and name ne# ) 3 .6 3 .6 0 .0 arom s assign i resid 36 and name nα*) (resiα a- and name nd#) 3 .6 3 .6 0 .0 1 arom s assign 'resid 36 and name hd#) (resid 37 and name he#) 3 .6 3 .6 0 .0 arom s assign resid 37 and name na) (resiα 37 and name nd#) 2 .8 2 .8 0 .0 arom i assign resiα 2 — and name hd#) (resiα 90 and name hb) c .5 5 .5 0 .0 arom m assign resiα 37 and name nd#) (resiα 90 and name hg2#) 5 .5 5 .5 0 .0 arom m assign (resid 37 and name nd#) (resid 90 ana name hd#) 3 .6 3 .6 0 .0 arom m assign resid 37 and name hd#) (resid 91 and name hb#) 3 .6 3 .6 0 .0 arom m assign resid 37 and name hd#) resid 91 and name hd#) 2 .8 2 .8 0 .0 arom m assign resid 3" and name hα#) resiα 91 and name ne#) 3 .6 3 6 0 .0 arom m assign resiα 37 and name ne#) resiα 91 and name hd#) 5 .5 5 5 0 0 arom m assign resiα 37 and name ne#) resiα 91 ana name he#) 5 .5 5 5 0 0 arom m assign resid 91 and name hn) resiα 91 and name hd#) 3 6 3 6 0 0 arom I assign resid 91 and name hd# ) resid 92 and name hn) 5 5 5 5 0 0 arom s assign resid 2 and name nd ) resiα 86 ana name hd#) 5 5 5 5 0 0 eth 1 assign resid 5 ana name nbif) resid 86 and name hd*) 5 5 5 5 0 0 metn 1 assign resid 17 and name rn) resid 90 and name hd#) 4 1 4 1 0 0 meth_l assign resid _" 1 and name na*) resiα 90 and name hα#) 5 5 5 5 0 0 meth 1 assign resiα 29 and name ho#) resid 90 and name hd#) 5 5 5 5 0 0 meth 1 assign resid 29 and name ng#) resid 90 and name hd#) 5 5 5 5 0 0 meth_l assign resid 29 and name hα#) resiα 90 and name hd#) 5 5 5 5 0 0 meth 1 assign resid 29 and name he#) resid 90 and name hα#) 5 5 5 5 0 0 meth 1 assign resid 31 and name hn) resid 90 and name hd#) 5 5 5 5 0 0 meth 1 assign resid -31 and name ha) resid 90 and name hg2#) 5 5 5 5 0 0 meth 1 assign resiα 31 and name na) resiα 90 and name hd#) 5 5 5 5 0 0 meth 1 assign resid 32 and name ha) resiα 90 and name hg2#) 5 5 5 5 0 0 meth 1 assign resid 32 ana name nb#) resid 90 and name hg2#) 5 5 5 5 0 0 meth_l assign resiα 32 and name hg) resiα 90 ana name hg2#) c 5 5 n 0 meth 1 assign resid 40 and name nn) resiα 40 and name ne#) 5 5 5 5 0 0 metn i assign resid _ι 7 and name ha) resid 43 and name ng2#) 5 5 5 5 0 0 meth s assign resid 47 and name hoi) resiα 74 and name hd#) 5 5 5 5 0 0 meth 1 assign resid 47 ana name hg#) resiα 74 and name hd#) 5 5 5 5 0 0 meth 1 assign ( resid 43 and name hn) resid 48 and name hg2#) 3 6 3 6 0 0 meth l assign ( resid 48 and name hn) resid 74 and name hd#) 5 5 5 5 0 0 meth 1 assign ( resid 48 and name ha) resid 74 ana name hd#) 3 6 3. 6 0 0 meth 1 assign ( resid 48 and name hg2#) resid 49 and name hn) 5 5 5. 5 0 0 meth s assign ( resid 48 and name ng2#) ( resid 50 and name hn) 3 6 3. 6 0 0 ircet m assign ( resid 48 and name hg2#) ( resiα 50 and name hbl) 5 5 5. 5 0. 0 meth m assign ( resid 43 and name hg2#) ( resiα 51 and name hn) 3 1 3. 1 0. 0 metn m assign ( resid 48 and name hg2#) ( resid 51 and name ha) 3. 6 3. 6 0. 0 meth -n assign ( resid 48 and name hg2#) ( resid 51 and name hb#) 3. 6 3. 6 0. 0 metn m assign < resid 43 ana name hg2i) ( resiα 51 and name hg#) 5. 5 5. 5 0. 0 meth m assign ' resid 43 and name hg2#ι ( resiα 74 and name nd#) 5. 5 5. 5 0. 0 meth 1 assign resid 49 and name n) ( resiα ^4 and name nd*) 5. 5 5. 5 0. 0 me h_l assign ι resid 49 ana name ha) ' resiα 74 and name hd#) 5. 5 5. 5 0. 0 meth 1 assign resid 54 and name nn) res ic 4 and name hd#) 5. 5 5. -: 0. 0 meth 1 assign resid 54 and name na=f) ' resiα ana name hg2p) 5. c 5. 5 0. 0 m 1 assign resid 54 and name ha?) ' resiα 1 — and name nd#) 5. 5 5. 5 0. 0 meth 1 assign ^ = . ^-. 56 ana name nn ) ' resiα 60 and name no ) 5 5 5. 5 0. r * etn m assign resiα 56 ana name nn) v resiα " 4 and name hg2 ) 5. 5 5. 5 0. 0 meth _ assign ' resid 56 and name na) ' resiα 60 and name no#) o . 5 5. 5 0. 0 metn m assign resid 56 a a name na) ' resiα 90 and name hg2#) 5. 5 5. 5 0. 0 m o th 1 ass gn resiα 56 ana name nol resiα 60 ana name hb*ι 3. 6 3. 6 0. 0 meth assign res.a 57 ana name nn) resiα 6D ana name hb#) 3. 6 3. 6 0. C ' metn m - - assign iresiα - ~" ana name naj resiα 60 ana name no*) 5 .3 3 .5 ^ me h m assign ' resid ana name Γ.D- resid 60 ana me nb= - .3 3 .3 ^ ~ - ι etn m assign ' esiα 53 ana ame no?) 'resiα 59 ana name n 3 .6 3 .6 ^ . : retn s assign ' resiα 53 ana m0 no* resid 60 ana name 5 .5 5 .3 0 2 - rn r assign (resiα 59 ana name nn) 1 resid 60 ana name ho= 3 .5 5 .5 0 . c metn s assign (resiα 60 and name r.Ω≠ ) \ resiα 61 ana name hr. ; 3 .6 3 .6 0 meth s assign resid 6 J ana name nb*) ! τ "i 3 5i i 76 a •" τ^ o r.a , 3 .3 3 .3 J ~) retn 1 assign resid 0 ana name Γ.D*) iresiα ana name hoi) 5 .5 5 .5 . z retn 1 assign resi 61 and name no#) resiα — — na name hn> 3 .6 3 .6 Z metn _ assign resiα 60 ana name hb*) resid 77 ana name na / 5 .5 5 .5 c meth 1 assign resiα 60 and name hb#) ( resid 78 ana name hn; 5 .5 5 .5 ^ n meth 1 assign resid 60 ana name ho#) (resid 73 ana name hbl) 5 .5 5 .3 0 meth 1 assign resid 62 and name na) (resid 74 and name hg2*) 5 .5 5 .5 0 .0 meth 1 assign resiα 63 and name nn) (resid 74 ana name hg2#) 4 .1 4 1 n 0 meth_l assign resiα 63 and name n) (resid 74 ana name nα*) 3 .6 3 6 0 0 meth 1 assign resid 63 and name hg2#) 1 resid 64 ana name hn; 3 .6 3 6 0 0 metn s assign resiα 64 and name ha) (resid 74 and name hg2#) 5 .5 5 5 0 0 metn 1 assign resid 64 and name ha) (resid 74 ana name hd#) 5 .5 5 5 0 0 meth 1 assign resid 64 and name hbfr) (resid 74 and name hd#) 5 5 c 5 0 0 meth 1 assign resid 64 and name hg*) (resid 74 and name hd#) 5 5 5 5 0 0 meth 1 assign resiα 72 and name nn) (resid 72 and name he*) 3 6 3 6 0 0 meth 1 assign resiα - 2 and name nb*) (resid " ana name hα*) 5 5 5 5 0 meth m assign resiα - > 3 and name hn) 'resid 74 ana name hα#) 5 5 5 5 0 meth s assign resid "4 and name hn) (resid 74 and name hg2#) 3 6 3 6 0 0 metn 1 assign resid 74 and name ha) resid 74 ana name hg2#) 3 6 3 6 0 0 meth 1 assign resiα 74 and name ha) (resid 74 and name hd*) 3 6 3 6 0 0 meth 1 assign resid 74 and name ng2ff) (resid 75 ana name ha. 5 5 5 5 0 0 meth s assign resid ^5 and name ng2#) (resiα 76 and name hn) 3 6 3 6 0 0 meth_s assign resid ^9 and name nn) (resid 79 and name hg2 # ) 3 6 3 6 0 meth 1 assign resiα 79 and name hg2#) (resiα 80 and name hn) 3 6 3 6 0 0 meth s assign resid 35 and name na) (resid 86 ana name hα*) 5 5 5 0 0 meth s assign resid 86 and name hd#) (resid 87 and name hn) 5 5 5 5 0 c meth s assign resid 86 and name hd#) (resid 91 and name hb#) 5 5 5 5 0 0 meth 1 assign resid 36 and name hd#) (resid 92 ana name na) 5 5 5 5 0 0 meth 1 assign resid 37 and name ha) (resid 90 and name hd#) 5 5 5 5 0 0 meth m assign resid 37 and name hb*) iresid 90 ana name ng2#) 5 5 5 5 0 0 meth m assign resid 37 and name hb*l (resid 90 and name hd#) 5 5 5 5 0 0 meth m assign resid 39 and name hn) (resid 90 ana name hg2 ) 5 5 5 5 0 meth s assign resiα 90 and name nn (resid 90 ana name hg2 ) 3 6 3 6 0 0 metn 1 assign resiα 90 ana name na) resid 90 ana name hg2#) 3 6 3 6 0 metn 1 assign resid 90 and name ng2*) (resiα 91 ana name nn) 5 5 5 5 nu ; neth s assign resid 90 ana name hd# ) (resid 91 ana name nn) 5 5 5 5 c meth s assign ( resid 6 and name ha) (resid 6 ana name ng*) 2 8 2 8 0 0 dgn 1 assign ( resid 10 and name he#) (resid 19 and name hn) 3 6 3 6 0 0 dgnm 1 assign ( resid 10 and name he#) (resid 28 and name hn) 3 6 3 6 0 0 dgnm 1 assign ( resid 14 and name nn) (resid 14 and name hg#) 3 6 3 6 0 0 dgnm 1 assign ( resid 14 and name hb#) (resid 15 and name hn) 4 1 4 1 0 0 dgnm s assign ( resid 24 and name nn) (resid 24 and name ng ) 3 6 3. 6 0 0 c-gnm 1 assign resid 24 and name ha) (resid 24 and name hg ) 2 3 2 8 0 0 αgnm 1 assign ( resid 24 and name hg?) (resid 25 and name nn) 5 5 5 c 0 dgnm s assign ( resid 25 and name hni (resid 25 and name nα*) 5 5 5 5 0 0 dgnm 1 assign ( resid 26 and name hn) (resid 26 and name hb ) 2 8 2. 8 0 c αgnm 1 assign ( resid 29 and name hb?) (resid 29 ana name nd#) 3 6 3 6 0 0 dgnm 1 assign ' resiα 29 and name ho# iresid 30 and name hn) 3 6 3 6 0 0 dgnm s assign ' a5i_ri 33 and name hn) 'resiα 47 ana name ng*) 5 5 5. c dαn 1 assign ( resiα 35 and name hn) (resiα 35 ana name hg*) 3 6 3. 6 αgnm 1 assign ' -03 . ci 25 and name na (resid 35 ana name hg*) 3. 6 3. 6 αgnm _ assign ssic 35 and name hg* ) ( resiα 36 ana name hn) 3. 6 3. 6 0 αgnm s assign resiα 35 ana name hg*) (resid 42 ana name n ; 5. 5 5. 5 0 dgnm 1 assign ' resiα 35 and name ? resid 44 ana name ha^ 6 3. 6 0 αgnm 1 assign ( resid 35 and name hg ) ( esid 44 ana name np?) 3. 6 3. 6 0. αgnm 1 assign ( resiα 36 and name hn) ( resid 36 ana name ng*) 3. 6 3. 6 0. 0 αgnm 1 assign ( res1a 36 and name ha) resid 36 and name ng*) 2. 8 2. 8 0 dgnm 1 assign \ vog ^ 36 and name no? 1 (resiα 37 and name hn, 5 3 0. αgnm s assign resiα 36 and name ha- ' (resid 37 a a name hr 1 3. 6 3. 6 ^ αgnm s assign > resια 26 ana name h *; { resid 40 ana name n.n .5 3.5 0 1 dgnm assign ( resid and name na! ( resid 37 and name ng*/ 3 .6 .6 0 .0 1 αgnm 1 assign resια ~ ~~ and name hg* (resid 33 and name hn) 4 . _ 4 ι_ 0 n 1 dgnm s assign (resid - 3 ana name nn! (resid 40 and name ng* ) 3 .6 3 .6 0 .0 1 αgnm 1 assign (resiα 4 Z ana name nn) (resiα 40 and name nα* ) 5 .5 5 .5 0 3 ! dgnm 1 assign (resiα 44 and name ha) (resid 45 ana name ng=) 3 .6 3 6 0 0 1 αgnm s assign resid 4 z ana name ng?) (resid 46 and name hn ) 3 6 3 6 0 0 1 dgnm s assign resid 4 " and name ng*; tres d 48 ana name nn ) 5 5 5 5 0 0 1 dgnm s assign resid and name hn! (resid 51 and name ng*) 3 6 3 6 0 0 ! αgnm m assign resid 49 ana name hn) (resid 64 ana name hb#) 3 3 5 0 0 1 dgnm_l assign resid 49 and name hn) (resid 64 and name hg?) 5 5 5 5 0 0 1 dgnm 1 assign resid 31 and name hn) (resid 51 ana name ng*) 3 6 3 6 0 0 1 dgnm 1 assign resid 31 ana name hg*) (resid 52 and name hd22) 5 5 5 5 0 0 ! gnm s assign resid 61 ana name hn) (resid 61 ana name hb*) 2 8 2 8 0 0 ! αgnm 1 assign resid 61 and name hb*) (resid 62 and name hn) 3 6 3 6 0 0 ! dgnm s assign resid 64 and name hn) (resid 64 and name hb#) 2 8 2 8 0 0 ! dgnm 1 assign resiα 64 and name hn) (resid 64 and name hg*) 2 3 2 8 0 0 ! dgnm_ι assign resid 64 and name hn) (resid 65 and name hg*) 2 8 2 8 0 0 ! dgnm s assign resid 64 and name ha) (resid 64 and name hg#) 3 6 3 6 0 0 ! dgnm_ι assign resid 64 and name hb#) (resid 74 and name ha) 3 6 3 6 0 0 ! dgnm 1 assign resiα 65 and name hn) (resid 65 and name ng*) 3 6 3 6 0 0 ! dgnm 1 assign resid 69 and name hb*) (resid 70 ana name hn) 2 8 2 8 0 0 1 dgnm_s assign resid / _ and name ha#) (resid 72 and name hn) 2 6 2 6 0 0 ! dgnm s assign resid 2 ana name hn; (resid 72 ana name hd#) 3 6 3 6 0 0 1 dgnm 1 assign resid 73 and name hn) (resid 73 and name hb#) 2 3 2 8 0 0 ! dgnm 1 assign resid 73 and name hb#) (resid 74 and name hn) 3 6 3 6 0 0 ! dgnm s assign resid 80 and name hn) (resid 80 ana name hb? ; 2 3 2 8 0 0 ! dgnm 1 assign ( resid 30 and name no*) (resid 81 and name hd*) 2. 3 2. 8 0 0 ! dgnm s assign resid SO and name ho* ; (resid 83 and name hn) 3 6 3 6 0 0 ! dgnm m assign resid SO ana name hb#) (resid 83 and name hb#) 2. 3 2 8 0 0 ! dgnm m assign resid 34 and name ha) (resid 85 and name hg#) 3. 6 3 6 0 0 ! dgnm s assign ( resid 84 and name ha) (resid 85 and name hd#) 2. 8 2. 8 0. 0 ! dgnm s assign ( resid 85 and name hg#) (resid 89 and name hn) 5. 5 5. 5 0. 0 ! dgnm m assign ( resid 86 and name hb#) (resid 87 and name hn) 3. 6 3. 6 0. 0 ! dgnm s assign ( resid 86 and name hb*) (resid 91 and name hb#) 3. 6 3. 6 0. 0 ! dgnm 1 class nsam assign resid 3 and name ha) (resid 8 and name hα*) 3 6 3 .6 0 0 'metn 1 assign resiα and name hg*) (resid 20 and name hα*) 5 5 5 .5 0 0 'meth _ assign resid 3 and name hg*ι (resid 20 and name he) 5 5 5 5 0 0 1 meth ^ assign resid —3 and name ha) (resid 31 and name hdl = ) 3 6 3 6 0 0 !meth 1 assign resid 15 and name hb#) (resid 31 and name hdl*) 5 5 5 5 0 0 !meth " l assign resid 16 and name hn) (resid 31 and name hdl#) 5 5 5 5 0 0 !meth " l assign resid 16 and name ha) (resid 31 and name hd#) 5 5 5 5 0 0 !meth_ " l assign resid 17 and name hn) (resid 31 and name hd2*) 4 1 4 1 0 0 !meth " l assign resid T_7 and name ha#) (resid 31 and name hd2#) 5 5 5 5 0 0 'meth_ " l assign resid 19 and name hz) (resid 22 and name hd#) 3 6 3 6 0 0 !πteth_ m assign resid 22 and name hd#) (resid 23 and name hn) 5 5 5 5 0 0 !meth s assign resiα 30 and name ha) (resid 31 and name hd#) 5 5 5 5 0 0 'meth s assign resid 31 and name ha) (resid 31 and name hd24) 3 6 3 6 0 0 ' meth 1 assign resid 31 and name hd2#) (resid 87 and name hb#) 5 5 5 5 0 0 !metn 1 assign resid 31 and name hd2#) (resid 87 and name nz) 3 6 3 6 0 0 ! meth _ assign resid 3 _ and name hd2#) (resid 90 and name hg2*) 5 5 5 5 0 0 !meth 1 assign resiα 31 and name hd2ff) (resid 90 and name hd?) 5 5 5 5 0 0 !metn 1 assign resid 32 and name ha) (resid 32 and name nd2#) 3 6 3 6 0 0 !meth 1 assign resid 32 and name hd2#) ( resid 33 and name nn) 3 6 3 6 0 0 Imeth s assign resid 22 and name hd2 ) (resiα 55 and name na*) 5 5 5 5 n 0 'meth " ι_ assign resid 32 and name hα2#) { resid 56 ana name hn! 5 5 5 5 0 0 'meth _ assign resid 3 i- ana name hd2*) (resid 56 ana name na) 5 5 3 5 0 0 1 metn assign resid 32 ana name hd2?) (resid 74 and name hb) 5 5 5 5 0 0 1 meth assign resid 32 and name hd2#) (resid 74 and name ng2*! 5 5 5 5 0 0 Imeth 1 assign resid 32 ana name hd2#) (resid 90 and name ha; c 3 5 5 0 0 ' metn assign resid 32 ana name nα2*) (resiα 90 ana name ng2=) 3 6 6 0 0 ' meth _ assign resid 32 and name ndl*) ' resid 88 ana name ho=ι 5 c 5 -; 0 ! metn _ assign resid 35 ana name hn) iresid 42 anc name hg2 * ) 3.5 5.3 0.0 metn 1 assign resid 33 ana name nb2) resiα 42 ana name hgl*, 5 .5 5 .3 0 .0 • »eta 1 assign resid and name hg?) resiα 42 a- a name ng2*; 5 5 5 0 .0 metn 1 assign resid 7 — a"α name hn) resiα 42 ana name hgl* / 5 .5 5 .5 0 retn 1 assign resid 3 " ana name hn) resiα 42 ana name ng2* v 5 .5 5 . ~> 0 retn J. assign iresid 3 ^ ana name na) (resiα 42 ana name ng2*) 5 .5 3 .5 0 .0 metn 1 -. — assign resid a a name hD#) resiα 42 ana name ngl*) 5 .5 5 .5 0 .0 metn _ assign resid and name no#ι resiα 42 ana name ng2*ι 5 .5 5 5 0 .0 retn 1 assiσn resid 3 " ana name hg*) resiα 42 ana name nαl*) 5 .5 5 3 0 0 metn 1 assign resiα 37 ana name hg#) resiα 42 and name hg2*) 5 .3 5 .5 0 0 metn 1 assign 'resid ^0 ana name hn) resiα 42 ana name ngl*) ς .5 3 5 0 0 metn assign resiα 40 ana name hg*) resiα 42 and name hgl*) 5 .5 5 5 0 0 meth assign resid 41 and name na) (resid 42 ana name hgl*) .5 5 5 0 0 meth s assign 'resid 41 and name ha) (resid 42 ana name ng2*) 5 5 5 5 0 0 meth s assign 'resid 4 _. and name hb#) 'resid 42 ana name ng*) 5 5 5 5 0 0 meth s assiσn (resid 42 and name hgl#) (resid 43 ana name hn) 3 6 3 6 0 0 meth s assign (resid 42 and name hg2#) (resiα 43 ana name hn) 3 6 3 6 0 0 meth s assign (resiα 5 and name ha) (resid 20 and name hb#) 3 6 3 6 0 0 nsam 1 assign (resid 7 and name ha) (resid 20 and name hb#) 3 6 3 6 0 0 nsam_l assign lϊesid —> ana name ha) iresid 20 and name hd#) 3 6 3 6 0 0 nsam 1 assign resid 10 and name ha) ' resid 10 ana name ng*) 3 6 3 6 0 0 nsam i assign resiα 12 ana name ha) ires α 13 ana name nd*) 2 3 2 3 0 0 nsam s assign resiα 12 and name nb#) resiα 13 ana name nα*) 3 6 3 6 0 0 nsam s assign »resιd 12 ana name hb#) (resiα 23 ana name hb#) 3 6 3 6 0 0 nsam 1 assign (resid 13 ana name hb2) (resid 14 and name nn) 3 6 3 6 0 0 sing s assign 'resid 13 and name hg#) (resid 16 ana name hb*) 3 6 3 6 0 0 nsam m assign resid 13 and name hd#) 'resid 28 ana name no*! 3 6 3 6 0 0 nsam 1 assign (resid 15 and name no2 ) (resid 41 and name ho2) 3 6 3 6 0 0 sing 1 assign 'resid 16 ana name na) (resiα 30 and name hb#) 3 6 3 6 0 0 nsam 1 assign 'resid 16 ana name hb#) (resid 30 and name na) 3 6 3 6 0 0 nsam 1 assign 'resid 16 and name hb#) (resid 31 and name hn) 3 6 3 6 0 0 nsam 1 assign (resid 17 and name na#) (resid 87 and name hz) 3 6 3 6 0 0 nsam 1 assign (resid 20 and name ha) (resid 20 ana name hg#) 3 6 3 6 0 0 nsam I assign (resid 20 and name ha) (resid 26 and name hg*) 3 6 3 6 0 0 nsam 1 assign (resid 20 and name hb*) (resid 20 and name hα*) 3 6 3 6 0 0 nsam i assign (resid 20 and name hg*) (resid 21 and name hn) 3 6 3 6 0 0 nsam s assign (resid 20 and name hg*) 'resiα 24 ana name ha) 3 6 3 6 0 0 nsam m assign resid 21 and name hb*) resiα 22 ana name nn ) 5 5 5 5 0 0 nsam s assign resid 21 and name ho*) 'resiα 23 ana name nn) 5 5 5 5 0 0 nsam m assign 'resid 22 ana name nb2) resiα 23 ana name nn) 3 6 3 6 0 0 smg s assiσn 'resid 24 and name hb*) (resiα 25 ana name nn) 5 5 5 5 0 0 nsam s assign 'resid 25 ana name hn) (resiα 25 ana name ng#) 3 6 3 6 0 0 nsam i assign (resid 25 and name hg#) (resiα 26 ana name hn) 3 6 3 6 0 0 nsam s assign (resid 26 and name hn) (resid 26 and name hg*) 3 6 3 6 0 0 nsam I assign (resid 26 and name ha) (resid 26 ana name hg#) 3 6 3 6 0 0 nsam I assign (resid 27 ana name ha) (resid 27 and name hg*) 2 8 2 8 0 0 nsam l assign (resid 27 and name hb*) (resiα 28 and name hn) 2 8 2 3 0 0 nsam s assign (resid 27 ana name hg*) (resid 28 and name hn) 2 3 2 3 0 0 rrsam s assign (resid 28 and name hn) (resiα 23 and name hb#) 2 3 2 8 0 0 nsam i assign (resid 29 and name ng#) (resid 30 and name hn) 2 8 2 3 0 0 nsam s assign (resid 29 and name hd*) (resiα 90 and name hb) 3 6 3 6 0 0 nsam 1 assign (resid 30 and name hn) (resid 30 ana name hb*) 2 3 2 8 0 0 nsam i assign resid 30 and name hbl) ' resiα — 1 ana name hn) 3 6 3 6 0 0 smg s assign (resid 30 ana name hb#) resiα 34 ana name hn) 5 5 5 5 0 0 nsam ass ign resid 30 ana name nb2) resiα 24 ana name hb2) 3 6 3 6 0 0 smg m assign 'resid 30 and name hb*) resiα 3 ana name ha) 3 6 3 6 0 0 nsam 1 assign resid ana name hn) resiα 21 ana name hb*) 3 6 3 6 0 0 nsam i assi n resid 31 and name hn) res-.α 34 ana name hb2) "^ 6 3 6 0 0 smg assign resiα 32 ana name hbl) resiα ana name nn) 3 6 3 6 0 0 smg s assign resid 33 ana name ha, resiα - anc name hb=) 3 6 3 6 0 0 nsam 1 assign resid 33 ana name ha) resiα — 7 ana name hbl) 3. 6 3 6 0 0 smg 1 assign resid 33 ana name hb2, ' resiα ana name nn) 5. 5 5 _ 5 0 0 smg s assign ' resiα 34 ana name na) resiα 44 ana name hb*) 3. 6 3. 6 0 0 nsam 1 assign resid 34 ana name nol resiα 25 ana name nn) 4. 1 ά 1 0 n smg s assign resid 35 ana name no* / resiα 33 ana name nα=) 2. 8 2 . 3 0. 0 nsam ■ 8- assign resiα 3s ana name o2 ; resiα na name nn; 2 .6 3 .6 assign resiα 33 ana name no* resiα 44 ana name nb*) - . .0 ^ assign resiα 33 ana name α* resiα 44 a a name i _ 0 .0 -. assign ^ ι - ana name ) re ≤ lα 40 ana name no* ) ^ assign resiα 23 and name na* ' resiα 39 ana name nn ; 2 .1 3 .1 assign esiα 33 and name a*) resiα 40 ana name nn; 3 .3 5 .5 1 assign resiα 29 ana name o* resiα 40 ana name nn) 3 .6 3 .6 — assign v resια 41 ana name r.Dl; resiα 42 ana name nn, .5 3 .6 assign (resiα 43 and name nn ' 'resiα 43 ana name ho*) 2 .3 2 .8 0 assign ' resiα 3 and name a) resiα 43 ana name ng*) 2 .3 2 .3 r assign 'resid 43 and name nb*l resid 44 ana name nn) 3 .6 3 .6 υ assign resiα 43 ana name ng?) ( resid 44 ana name hn) 3 .6 3 .6 0 assign ( resid 44 and name nn) ' resid 45 ana name nd*) 5 .5 5 .5 0 assign 'resiα 44 and name na) (resiα 45 ana name nd*) .3 2 .3 0 assign (resiα i ana name nD2) (resiα 46 ana name hn) 3 .6 3 .6 0 assign ' resiα 46 and name nn) ' resid 46 and name nb*) 3 .6 3 .6 0 assign (resiα 46 ana name hn) (resid 47 ana name hd#) 3 .6 3 .6 0 assign ,resιd 46 and name ha) (resid 47 and name hd*) 3 .6 3 .6 0 assign (resid 47 and name hb2) (resid 48 and name hn) 4 .1 4 .1 0 assign (resid 47 and name hb2) (resiα 74 and name ngl*) 3 .6 3 6 0 assign (resid 47 and name hg*) (resid / 4 and name hgl*) 3 6 3 6 0 assign resiα 43 ana name nn) ' resiα 34 and name ha*) 1 4 1 0 assign ' resiα 48 ana name hn) (resiα 74 and name ngl*) 5 5 5 5 0 assign (resid 49 ana name ha) (resiα 54 and name ha#) 3 6 3 6 0 assign (resid 4 a ana name a/ (resid 62 and name hbl) 5 5 5 5 0 assign iresid 50 and name nol ' (resiα 51 ana name hn) 3 6 3 6 Λ assign resid 53 and name no*) (resid 56 ana name hn) 5 5 5 5 0 assign iresid 53 ana name nol; (resid 58 ana name ha) 3 6 3 6 0 assign (resid 53 and name nd22) (resid 56 and name hn) 5 5 5 5 u assign (resid 53 ana name hd21) (resid 60 and name hn) 5 5 c 5 0 assign (resid 54 and name ha*) (resid 55 and name hn) 3 6 3 6 0 assign (resid 54 and name na*) (resid 56 and name hn) 3 6 3 6 0 assign (resid 55 and name na*) (resid 56 and name hn) 3 6 3 6 0 assign (resid 56 and name hbl) (resid 57 and name hn) 4 1 4 1 0 assign (resid 56 and name nb2) (resid 62 ana name nn) 5 5 5 5 0 assign (resiα 56 and name no2) (resid 62 and name ha) 3 6 3 6 0 assign s resιd 57 and name ho*) (resid 58 and name nn) 3 6 3 6 0 assign resiα 57 and name Γ.D*) (resid 59 and name nn) 1 4 1 0 assign 'resid 57 and name no*! ι resid 60 and name nn) 4 1 4 -L 0 assign 'resid 5"" and name ho*) 'resiα 91 ana name na) 2 8 2 3 0 assign 'resiα 57 and name nb* 1 resια 92 and name nn) 5 5 5 5 0 assign (resiα 53 and name nn) (resid 59 and name hb*) > 5 5 5 0 assign (resid 59 and name hb*) (resid 80 ana name ng#) 3. 6 3 6 0 assign (resid 59 and name hb#) (resid 60 and name hn) 3. 6 3. 6 0 assign (resid 60 and name ha) (resid 73 and name hb#) 3. 6 3. 6 0 assign (resid 61 and name nn) (resid 61 and name hg*) 3. 6 3. 6 0 assign (resid 65 and name hn) (resid 65 and name hb*) 2. 8 2. 8 0 assign iresiα 66 and name nn ) v resid 66 and name no*) 2. 8 2. 3 0 assign 'resid 66 and name hb*) 'resid 67 and name nn) 3. 6 3 6 0 assign (resid 67 and name hb*) (resiα 73 and name hb*) 3. 6 3. 6 0 assign f resid 68 and name ha?) iresid 69 and name hn) 3. 6 3. 6 0 assign (resid ^2 ana name hn) ( resiα 72 and name hg*) 3. 6 3. 6 0 assign (resid 74 and name hn) iresiα 74 and name ngl*) 3. 6 3. 6 0 assign (resiα 74 and name na) iresid 74 ana name hgl*) 3 _ 6 3. 6 0 assign 'resiα "J o ana name hn) (resid 76 ana name hb*) 2. 8 2. 3 0 assign ι resid 76 and name hoi) resid 77 ana name hn) 5. 5 5. 5 0 assign ι.resιd " 6 ana name hbl 'resid 39 ana name na*) 2. 6 3. 6 r- assign v resiα 73 ana name hbl) 'resiα "■ 9 ana name nn) 5. 5 5. 5 υ assign iresiα 73 ana name no*! (resiα 92 a a name nn* , 3. 6 3. 6 0 assign 1 esid -3 ana name ho*^ resiα 30 ana name hn) 3. 3 3. 5 c assign resiα 78 ana name hb* j ( resiα 83 ana name nc=) 3. 6 3. 6 0 assign (resid 80 and name nn) resiα SO ana name ng*) 3. 6 3. 6 0 assign ■ "ώg ι H SO ana name nr ) ,resια 31 and name nα*) 5. 5 5. 5 c assign 'resiα 30 ana name nr.i 'resiα 33 ana name nb* / 3. 6 3. 6 0 assign .resid 30 ana name na; ' resid S 0 ana name hg?) 6 3. 6 0 assign (resid 3 C and name n ) iresid 81 ana name hd * ! i..3 2.3 .nsam s assign ( resid -3 ^ and name hg* j (resi 31 and name nd f) 3 .6 3 .6 ^ .nsam s assign (resid 31 and h 2) ■ resid 82 and name r.~ 3 .6 3 .6 G c sing s assign (resiα 31 and name hd* ) (resid 32 and name h 3 .3 3 .3 u sam s assign (resid 82 and name hb*) (resid 33 and name hn 4 .6 3 .6 -nsam ≤ assign (resid 33 — and name ha) (resid 89 and name a T ; 3 .6 3 .6 G Λ nsam m assign . resiα 55 and name hb* ) ( resid 83 and name a 3 .6 3 .6 0 nsam m ass gn (resid 55 and name hb*) (resid 89 ana name hn 3 .6 7 .6 -3 nsam m assign ( resid 55 and name hb*) ( resid 36 ana name hn 3 .6 3 .6 n J nsam s assign (resid 36 and name hn) , resid 39 ana name ÷) 5 .5 5 .5 J nsam m assign (resid 57 and name hb*) (resid 90 ana name hn 3 .6 3 .6 u 0 nsam m assign (resid 37 and name hb*) (resid 90 and name ng .*! 5 c c .5 0 nsam m assign (resid 39 and name ha*) (resid 90 and name n 3 .6 3 .6 0 nsam s assign resid 90 and name hgl*; (resid 91 and name hn ^ 1 4 1 0 0 nsam s class si -ng assign resid 2 and name hn) (resid 3 and name hn 5 5 5 5 0 0 sing s assign resid 4 and name hn) (resid 5 and name hn 5 5 5 5 0 0 sing_s assign resid 5 and name hn) (resid 6 and name hn 3 6 3 6 0 0 sing s assign resid 3 and name ha) (resid 20 and name hn 5 5 q 5 0 0 sing 1 assign resid 6 and name hn) (resiα 7 and name hn 5 5 5 0 0 smg s assign resiα "* and name hn) .resid 3 and name hn 5 5 5 5 0 0 sing s assign resid 3 and name hn) (resid 9 and name hn 5 5 5 5 0 0 sing s assign resid 9 and name hn) ( resid 10 and name h 3 6 3 6 0 0 sing_s assign resid 1-3 and name ha) (resid 14 and name hn 2 6 2 6 0 0 sing s assign resid 14 and name hn ) (resid 15 and name hn 5 5 5 5 0 0 sing s assign resid 14 and name ha) (resid 15 and name hn 2 6 2 6 0 0 sing m assign resid 15 and name hn) (resid 16 and name hn 3 6 3 6 0 o sing s assign resid 13 and name ha) (resid 16 and name hn 3 6 3 6 0 0 sing s assign resid 15 and name ha) (resid 31 and name hg) 3 6 3 6 0 0 sing 1 assign resid 16 and name hn) (resid 17 and name hn) 5 5 5 c 0 0 sing s assign resid 16 and name hn) (resid 31 and name hn) 5 5 5 5 0 0 sing_l assign resid 16 and name hn) (resid 31 and name hg) 5 5 5 5 0 0 sing 1 assign resid 16 and name ha) (resid 30 and name ha) 2 3 2 8 0 0 sing 1 assign resid 16 and name ha) (resid 31 and name hn) 3 6 3 6 0 0 sing 1 assign resid 16 and name ha) (resid 31 and name hg) 3 6 3 6 0 0 smg 1 assign resid -_ and name hn) (resid 31 and name hn) 5 5 0 0 s g 1 assign resid 13 and name ha) (resid 19 ana name hn! 2 6 2 6 0. smg s assign resid 13 and name ha; (resid 28 and name na : 2 3 2 8 0. smg 1 assign resid 19 and name hn) (resid 20 and name hn! 5 5 3 5 0. Q sing s assign resid 19 and name hn) (resid 21 and name hd2) 5 5 5 5 0. 0 s g m assign resid 19 and name hn) (resid 28 and name ha) 5 5 5 5 0. 0 sing 1 assign resid 19 and name ha) (rεsid 20 and name hn) 3 1 3 1 0. 0 sing s assign resid 19 and name hz) (resid 60 and name hn) 5 5 5 5 0. 0 sing_l assign resid 20 and name ha) (resid 21 and name hn) 3 1 3 1 0. 0 sing s assign resid 20 and name ha) (resid 26 and name ha) 3 6 3 6 0. 0 sing 1 assign resid 21 and name hn) ( resid 21 and name hd2 ) 3 6 3 6 0. 3 s-ing i assign resid 21 and name hn) (resid 25 and name hn) 5 5 5 5 0. 0 sing m assign resid 21 and name ha) (resid 22 and name hn) 3 6 3 6 0. 0 sing s assign resid 21 and name ha) (resid 23 and name hn) 3 6 3 6 0. 0 sing m assign resid 22 and name hn) (resid 22 and name hg) 5 5 5 0. 0 sing i assign resid 22 and name hn) ( resid 23 and name hn) -; 5 5 5 0. 0 sing s assign resid 22 and name hn ) (resid 25 and name hn) 5 5 5 5 0. 0 smg m assign resid 22 and name ha) (resid 23 and name hn) 4 1 τ_ 0. sing s assign resid 22 and name ha) (rεsid 24 and name hn) 4 4 1 n 0 sing m assign resid 22 and name hg) (rεsid 23 and name hn, 5 5 c 5 0. 0 smg s assign resid 23 and name } (resid 24 and name hn ) 1 4 1 c. c smg s assign resid 22 and name hn) (resid 24 and name ha) 5 5 5 5 0. ^ sing s assi n resid 23 and n^- e ha) (resid 24 and name h ) 4 1 J 0. 0 sing s assign resid 23 and name ha) iresid 25 and name h ) 5 5 5 5 0. smg assign ( resiα 24 and name ha) (res ' id 25 and name hn) 5. 5 5. 5 0. 0 sing s assign ι rεsiα 25 and name hn) ( resid 26 and name hn) 3. 5 3 0. smg s assign ( resid 25 and name ha ) ( resid 26 and name n ) 2. 6 6 0. s g s assign ( resid 26 and name ) ' resid 27 ana name hn) 5. 5. 5 0. smg s assian resid 26 ana name ha 'resiα 2 " " ana name nn/ 3.6 3.6 0.0 s g s assign resiα _ ana name ra ^^Sl^ 23 ana name r ; /_ .6 2 .6 0 .0 smg s assign resiα 2B ana "ame a 'resiα 29 ana name nn 3 . _ 3 0 smg s assign resid Z 9 ana r.ar"e na resid 30 ana name n 7 .6 2 0 smg s assign i resiα ar α name nn. resiα 21 ana name n) 5 .5 5 .5 0 smg s assign resid ana name ha, (resiα 31 ana name nn, 3 .1 2 . i 0 sing s assign resid --._ ana name ' resiα 31 ana name ng; 3 .6 3 .6 0 sing _ assign v resid j ----. a a name hn (resiα 32 ana name nr 5 .5 5 .5 0 .0 s ng s assign resiα ana name na, iresiα 32 ana name nn ) 3 . _ .1 0 0 smg s assign ( resiα ana name na) iresid 31 and name ng) 3 .6 3 .6 0 0 smg assign resid 32 ana name ha. (resid 32 ana name hg! 3 .6 3 .6 0 0 smg i assign ' resid 32 and name ha) (resid 33 ana name nn) 2 6 2 .6 0 0 smg s assign resiα 32 ana name ha) (resid 34 and name hn, 3 6 3 6 0 0 smg m assign ( resid - ana name hn) (resid 34 and name nn) 3 6 3 6 0 0 smg s assign resiα 33 and name ha) (resid 34 ana name nn) 3 1 3 1 0 0 smg s assign (resiα 34 and name hn) (resiα 35 and name hn) 5 5 5 .5 0 0 smg s assign (resid 34 and name hn) (resid 44 and name hn) 5 5 5 .5 0 0 sιng_i assign resid 34 and name ha) (resid 35 and name hn) 3 1 3 1 0 0 sing s assign resid 34 and name ha) (resid 43 and name ha) 2 8 2 8 0 0 smg 1 assign resid 34 and name ha) (resid 44 and name hn) 3 1 3 1 0 0 smg 1 assign resid 35 and name hn (resid 36 ana name hn) 5 5 5 5 0 0 smg s assign resid 25 ana name nr.i resid 41 and name ha ; c 5 5 5 0 0 smg 1 assign resid 35 ana name nn) resid 42 ana name hn) 3 6 3 6 0 0 smg 1 assign resid 35 and name nn, (resid 43 and name ha) 5 5 5 5 0 0 smg 1 assign resid 35 and name hn) (resiα 44 and name hn) 5 5 5 c 0 0 sιng_l assign resid 35 and name nn) resiα 44 and name na) 5 5 5 5 0 0 s g 1 assign resid 35 ana name na) resid 36 and name nn) 6 ... 6 0 0 smg s assign resid 36 and name r. ) resid 37 ana name hn) 5 5 5 5 0 0 smg s assign resid 36 ana name na) resid 37 ana name hn) 2 6 2 6 0 0 smg s assign resid 36 and name ha) resid 41 and name ha) 2 8 2 3 0 0 smg 1 assign resid 36 and name ha) resiα 42 and name hn) 3 6 3 6 0 0 sing 1 assign resid 37 and name hn) resid 40 and name hn) 3 6 3 6 0 0 smg m assign resid 37 and name hn) resid 41 and name ha) 3 5 3 6 0 0 sing m assign resid 27 and name hn) resid 42 ana name hn) 5 5 5 5 0 0 smg_i assign resid 37 and name na) resiα 33 and name hn) 2 6 2 6 0 0 sing s assign resid 38 and name hn) resid 39 ana name hn) 5 5 5 5 0 0 smg s assign resid 39 and name hn; resid 40 and name nn) 3 6 3 6 0 0 smg s assign resid 40 and name hn) resid and name hn) 5 5 5 5 0 0 smg s assign resid and name ha) resid 41 ana name r.n' 2 6 2 6 0 0 smg s assign resid 41 and name nn) resiα 42 ana name hn) 5 5 5 0 0 smg s assign rεsid 41 and name na; resid 42 and name nn ) 6 2 6 0 0 s ng s assign resid 42 and name na) rεsid 43 and name nn) 2 6 2 6 0 0 smg s assign resid 42 and name ho) resid 43 ana name nn) 3 3 1 0 0 smg s assign resid 43 and name hn) resid 44 and name hn) 5 5 5 5 0 0 smg s assign resid 43 ana name ha) resid 44 and name nn) 2 6 2 6 0 0 s g s assign resid 44 and name ha) resid 46 and name hn) 3 6 3 6 0 0 smg m assign resid 45 and name ha) resid 46 and name hn) 3 6 3 6 0 0 smg s assign rεsid 46 ana name ha) resid 72 and name nn) 5 5 5 5 0 0 sα.ng_i assign rεsid 47 and name ha) resid 43 ana name hn) 2 6 2 6 0 0 smg s assign resid 48 ana name hn) resid 49 and name nn) 5 5 5 5 0 0 smg s assign resid 48 and name hn) resid 51 and name nn) c 5 5 5 0 0 smg assign resiα 48 and name ha) resid 49 and name hn) 3 1 3 1 0 0 smg s assign resid 43 ana name ha) resid 50 ana name nn) 5 _ 5 5 5 0 0 sing ro assign resid 43 and name ΠD) resid 49 ana name hn) 3. 6 3 6 0 0 smg s assign rεsid 43 ana name hb) resid 50 and name p ^ 5 5 5 5 c 0 smg r assign rεsiα 49 and name hn) resid 50 and name nn) 3. 6 3 6 0 0 smg s assign resid 49 and name nn 1 resiα ana name hn; 5. 5 5. 5 0. 0 s g assign resid 49 and name ha resiα 50 ana name nn! 3. 6 3. 6 0 smg s assign rεsid 49 ana name na / resiα 51 ana name hn, 1 0 smg ι assign resiα 49 ana name a resiα 53 ana name na' 3 _ 6 3 6 0 smg m assign resid 49 and name na resid 54 ana name hn 3 6 7 6 0 smg ... assign resiα 50 and name nn) ( resiα ana name nn) 3. 5 3. 6 0. 0 smg s assign resiα 51 ana name nn ' resiα 32 and name nn' 5. 3 5. 5 0. 3 smg s assign resid -: - and name hn i resiα 53 ana name nn Δ . 1 1 0 c smg n assign resiα 3 - ana name n * resiα ana name nr . 1 4 1 0 J smg ~ assign resid 51 ana name na j resid 52 and name nn ) 2. 3.1 0. c smg s assign resid 52 ana name h ) resiα 33 ana name nn ) 3 .6 3 .6 0 .0 s g s assign resid 52 and name na j r° ™ iα 53 ana name nn ) 3 .6 3 .6 0 ^ smg s ass gn resid 33 and name hn ) res.α 54 ana name nn ) n " v 1 smg s assign resid 53 ana name ha ) resiα 3 t ana name nn ) 3 .6 3 .6 0 ^ 1 sing s assign rεsid 33 ana name na ) resiα 56 and name nn ) 2 .3 2 .3 0 . c 1 smg m assign resiα 54 ana name nn ) resiα 55 an.α name nn ) 2 .6 3 .5 0 ' smg s assign iresid 33 and name hn ) resiα 56 ana namε hn ) 3 3 . _ 0 . ' smg s assign 'resiα 56 and name nn ) 'resiα 57 and namε nn ) 5 .5 c .5 0 .0 1 smg s assign resid 56 and name ha ; resid 57 and namε nn ) 3 .6 3 .6 0 .0 smg s as s ign (resid 57 ana name hn ) resiα 59 ana name hn ) 5 .5 5 .5 0 .0 ' sing m assign (resid 57 and namε nn ; resiα 60 and name nn 3 .5 5 .5 0 n ' smg m assign (resid 57 and name nn ) resiα 90 and name hn ) 5 .5 .5 0 .0 smg 1 assign (resid 57 and name nn ) 'resiα 90 and name ha ) 2 .6 2 .6 0 .0 1 smg 1 assign (resid 57 and name hn ) (resiα 91 and namε ha ) 5 .5 5 .5 0 .0 ' smg 1 assign (resid 57 and name hn ) (resid 91 ana name hn ) 5 .5 5 .5 0 .0 'smg 1 assign (resid 57 and name ha ) (resid 91 and name ha ) 3 .6 3 .6 0 .0 ' smg 1 assign (resid 58 and name hn ) (rεsid 59 and name hn ) 3 .6 3 .6 0 .0 sing s assign (resid 58 and name hn (resid 60 and name hn .5 5 .5 0 .0 smg m ass lgn (resid 58 and name ha ( resid 59 and name hn ) 3 .6 3 .6 0 .0 smg s assign rεsid 59 and name hn i resid 60 and name hn ) 3 6 3 .6 0 n smg s assign iresid 59 and namε na (resiα 60 ana name hn ) 3 6 3 .6 0 smg s assign (resid 60 and name nn (resiα 61 ana name hn 5 5 .5 0 0 sing s assign iresid 60 and name hn (resiα 78 and name ha 5 5 5 .5 0 0 smg 1 assign (resid 60 and name ha (resid 61 and name hn 2 6 2 6 0 c smg s assign (resid 60 and name ha ( resid 77 and name hn 5 5 5 5 0 smg 1 assign (resid 60 and name na (resid 78 and name ha 2 3 2 3 0 0 sing 1 assign (resid 60 and name a (resid 79 and name hn 3 6 3 6 0 0 smg_l assign (resiα 61 and name nn (resid 77 and name hn 3 6 3 6 0 0 smg 1 assign (resid 61 and name nn (resiα ^8 and name na 3 6 3 6 0 0 smg 1 assign (resid 61 and name hn (resiα 79 and name hn 5 5 5 5 0 0 sing 1 assign (resid 61 and name ha (resiα 62 and name hn 2 6 2 6 0 0 smg s assign (resid 62 and name na (resid 63 and name hn 2 6 2 6 0 c smg s assign (resid 62 and name ha (resid 76 and namε ha 2 8 2 8 0 0 smg 1 assign (resid 63 and name hn ' resiα 63 and name ho 3 6 3 6 0 sing l assign (resid 63 and name hn (resiα 64 and name hn 5 5 5 5 0 0 smg s assign (rεsiα 63 and name hn ( resiα 75 and name nn 3 6 3 6 0 G smg 1 assign (rεsid 63 and name hn (resiα 76 and namε ha 5 5 5 5 0 c smg 1 assign 'resid 63 and name ha iresiα 64 and namε nn 2 6 2 6 0 r smg s assign resid 63 and name no ,resια 64 and namε nn 3 1 3 ι_ 0 0 smg s ass ign (resid 64 and name ha iresid 74 ana name na 2 8 2 3 0 smg 1 assign resid 65 and name ha (resid 66 and name hn 2. 6 2 6 0 0 smg s assign (resid 66 and name hn) (resid 67 and namε hn 5. 5 5 5 0 0 smg s assign (resid 67 and name nn) (resid 63 and name hn 5. 5 5 5 0. 0 s g s assign (resid 67 and name ha) (resid 68 and name hn) 3. 6 3. 6 0. 0 smg s assign iresid 70 and name nn) (resid 71 and name hn) 3. 1 3. 1 0. c smg s assign (resid 70 and name ha) (resiα 71 and name hn) 3. 6 3. 6 0. 0 smg s assign (rεsid 71 and name nn) (resid " 2 and namε hn) 3. 6 3. 6 0.c sing s assign 'rεsid 72 and name hn) (resiα 73 and namε hn) 5. 5 5. 5 0. smg s assign (resid 72 and name ha; (resiα 73 and name hn) 2. 6 2. 6 0. 0 smg s assign (resid 73 and name hn) iresid 74 and name hn) 5. 5 5. 5 0. 0 smg s assign (resid 73 and name ha) ' resid " and name hn) 2. 6 2. 6 0. 0 sing s assiσn 'rεsid 74 and name hn) ' resid " and name hb) * 5 3. 0. 0 sing assign resid ~4 ana name ha) resid _ 5 and name nn) 2. 6 2. 6 0. smg s assign resiα 75 ana name hn) ' resiα 3 ana name ho) 3. 1 3. 1 0. n smg i assign iresid 75 ana name ha) ' -*~e s -> 6 and name nn) 2. 6 2. 6 0. c smg s assign 'resiα 75 and name hb) resiα 76 ana name nn) 3 _ 6 3. 6 0. 0 smg s assign > resιd 76 and name na) resiα " and name hn) 2. 6 2. 6 0. sing s assign 'resiα a a name nn) resiα 73 and name nn ' 5. 5 5. 5 0. smg s assig" i es d 7" and name ha / resiα ~3 and name nn) 2. 8 2. 3 0. C ' s_ng s assign rεsid 78 and name hn, ( resid 79 and name hn) 5. 5 5. 5 0. Q smg s assign (rεsid 78 ana name ha) rεsiα 79 and namε hn) 2. 6 2. 6 0. c s ng s assign ,rεsιd 19 ana name hn) (resic ~ > 9 and name ho) 7 6 3. 6 0. 0 s g _ assig n rεsid ^ 9 ana name hn) resiα SO and name hn 3. 6 3. 6 0. c sing s assi n resiα 79 ana name n r^ resiα 80 ana name nn , 5. c 5. 5 0. smg s assign 'resid 31 and name naj ' resid 32 and name hn, 3.6 3 6 0 smg s assign resiα _ and name na) 'resiα 33 ana name nn) 4 1 4 1 3 " smg m assign I resiα 52 and name nn; resid 83 ana name nn. 4 .1 4 X r 3 smg s assi n. resiα and name nai resiα 33 ana name nn/ I 1 1 r s g s assign resiα 53 and name nni resiα 64 ana name nn; 5 5 5 υ 3 smg s assign resid 53 ana name na) resid 34 ana name nn ) 6 2 6 c s g s assign og_ 54 and name nn ! resid 92 ana name hn) 5 5 3 5 0 sing_l assign and name resid 93 ana name hn 1 c 5 5 5 o smg_l assign resid 35 and name na; resid 86 and name nn) 2 6 2 6 0 smg s assign , resiα 35 and name na) resid 92 ana name ha) 1 3 2 8 0 smg 1 assign i resiα 56 and name nn) resid 86 and name ng) 3 6 3 6 0 smg i assign (resiα 35 and name hn) iresid 37 and name hn) 5 5 5 5 0 smg s assign (resiα 56 and name hn) resid 89 ana name hn) 4 1 4 1 0 0 smg m assign 'resid 36 and name nn) resid 90 and name hn) 5 5 5 5 0 0 smg m assign (resiα 36 and name nn) resid 91 and name hn) 3 6 3 6 0 0 smg_l assign (resiα 36 ana name ha) resid 87 and name hn) 3 6 3 6 0 0 smg s assign (resid 37 and name hn) resid 88 ana name hn) 4 1 4 1 0 0 smg_s assign (resid 37 and name na) resid 88 and name hn) 3 1 3 1 0 0 smg s assign 'rεsid 38 and name hn) resid 89 and name hn) 4 1 4 1 0 0 smg_s assign ( resiα 53 and name hn) resid 90 ana name hn) 5 5 5 5 0 0 smg m assign (rεsiα 33 and name na) resid 89 ana name hn) 4 1 4 1 0 0 s g s assign resiα S 9 ana name hn) resid 90 ana name hn) 3 6 3 6 0 0 smg s assign irεsid 90 and name hn) resid 90 and name nb) 3 6 3 6 0 sing l assign (resid 90 and name hn) resid 91 and name hn) 3 1 3 1 0 0 smg s assign (resid 90 and name nb) resid 91 and name hn) 5 5 5 5 0 0 sing s assign v resιd ana name nn) resid 92 and name hn) 5 5 5 5 0 0 smg s assign i resiα 91 and name ha) resid 92 and name hn) 3 1 3 1 0 0 smg s assign (rεsid 92 and name na) resid 93 and name hn) 3 1 3. τ_ 0 0 sing s class none assign (resiα 17 and name hn) resid 29 and name o) 2 0 0 3 0 3 hbnd assign (resid 17 and name n) resid 29 and name o) 3 3 0 3 0 3 h nd assign (resiα 17 and name o) rεsid 29 and name hn) 2 0 0 3 0 3 hbnd assign (resid 17 and name o) resid 29 ana name n) 3 3 0 3 0 3 hbnd assign (rεsiα 19 and name hn) rεsid 27 and name o) 2 0 0 3 0 3 hDnd assign (resid 19 and name n) resid 27 and name o) 3 3 0 3 0 3 nDnα assign ' resid 19 and name 0' resid 27 and name hn) 2 0 0 3 0 3 hbnd assign ,resια 19 and name o) resid 2 " ana name n) 3 3 0 3 0 3 nond assign ( eslα - : and name nn resid 42 and name o) 2 0 0 3 0 3 ncnd assign (resiα 35 and name n) resid 42 and name o) 3 3 0 3 0 3 hbnd assign (resid 35 and name o; resid 42 and namε hn) 2 0 0 3 0 3 nαnd assign (resiα 35 and name o) resid 42 and name n) 3 3 0 3 0 3 hbnd assign (resid 61 and name hn) resid 77 and name o) 2 0 0 3 0 3 hbnd assign (resid 61 and name n) resid 77 and name o) 3 3 0 3 0 3 hbnd assign (resid 61 and name o) resid i i and name nn) 2 0 0 3 0 3 hbnd assign (resid 61 and name o) resid 11 and name n) 3 3 0 3 0 3 nbnd assign (rεsid 63 and name hn) resid 75 and name o) 2 0 0 3 0 3 hbnd assign (resid 53 and name n) resid 75 and name o) 3 3 0 3 0 3 nbnd assign (resiα 63 and name o) resid 75 ana name hn) 2 0 0 3 0 3 hbnd assign (resiα 63 and name o) resid 75 and name n) 3 3 0 3 0 3 nDnd 1 dihedral angle restraints X-PLOR format 1 ' cni- 22 restrain .ts ' phi 25 restraints 1 psi 7 -: restraints ' cn - 2 restraints 1 1 <ENERG Y> <^NG E> <RANG E><EX? 0NΞNT chι-1 restraints assicn res .. ^ ana name n. res iα -. : anc lame ca ) 15 ana name co ) resiα 1 ; anc 1 . 0 - 50 . . assign (resid 19 ana na e n / i rεsid 19 ana name ca v resια 19 ana name co) (resiα 19 ana namε eg 1 .0 180 .0 ; o . c 2 assign ( esiα 22 ana name n ) (resiα 22 ana name ca (resiα 22 ana name CO) > resιd 22 ana name < -.~ 1 .0 -50 40 . 2 - assign ,resια 23 ana namε n , iresiα 23 ana name ca, i resiα 23 ana name CD) (resid 23 ana name cα 1 .0 60 .0 40 .0 assign (resiα 33 and name n ; ' resid 30 and name ca ,resιd 3 ana n^me CO; (resid 30 ana name sg 1 -60 40 .0 2 assign iresiα 31 ana name n ; 'resid 31 ana name ca (resiα 31 and name CD) ' resid 31 ana name eg .0 -60 .0 40 .0 assign (resid 32 ana name n ) (resid 32 and namε ca; (resid 32 and name CD) iresid 32 and name eg ' -_ .0 -60 .0 40 .0 2 assign (resid 33 and name n ) (resid 33 and name ca, (resid 33 and namε cb) (resid 33 and namε eg) 1 .0 -60 0 40 0 2 assign (resid 34 and name n ) (resid 34 and name ca, 'resiα 34 and name CD) (rεsid 34 ana name eg) 1 .0 -60 0 40 0 2 assign (resid 35 and name n ) (resid 35 and name ca) (resid 35 and name CD) (resid 35 and name eg) 1 .0 60 0 40 0 2 assign (resid 41 and name n ) (resid 41 and name ca) (resiα 41 and name CD) (rεsid 41 and name sg) 1 .0 -60 0 40 0 2 assign (rεsid 42 and name n ) (rεsid 42 and name ca) (rεsid 42 ana name cb; 'rεsid 42 and name cgli 1_ 0 -60 0 40 π 2 assign (resiα 49 ana name n ) (resid 49 ana name ca , (resid 49 and name cb) (resid 49 ana name sg) 1 0 -60 0 40 0 2 assign (resid 52 and name n ; (resid 52 and name ca ; (resid 52 and name cb) (resid 52 and name eg) 1 0 60 0 40 0 2 assign (resid 53 and name n ) (resid 53 ana name ca) (resid 53 and name CD) (resid 53 and name eg) 1 0 180 0 40 0 2 assign (resid 56 and name n ) (rεsid 56 and name ca) (resid 56 and name CD) (resid 56 and name sg) 1 0 -60 0 40 0 2 assign (resid 62 and name n ) (rεsid 62 and name ca) (resid 62 and name cb) (rεsid 62 and name sg) 1 0 180 0 40 0 2 assign (resid 74 and name n ) (resid 74 and name ca) (resid 74 and name cb) (resid 74 and name cgl) 1 0 -60 0 40 0 2 assign (rεsid 76 and namε n ) (resiα 76 and name ca) (rεsid 76 and name cb) (rεsid 76 and name sg; I 0 -60 0 40 0 2 assign (resid 78 and name n ) (resid 78 and name ca; (rεsid 78 and name CD) (resid 78 and name sg, 1 0 -60 0 40 0 2 assign (rεsid 87 and name n ) (rεsid 87 and name ca) (rεsid 37 ana name CD) (rεsid 37 ana name eg; 1 0 -60. 0 40 0 2 assign (resid 92 and namε n ) (resid 92 and name ca; (rεsid 92 ana name CD) (resid 92 and name sg) ]_ 0 -60. 0 40. 0 phi restraints assign (resid 14 and name resid 15 and name n ) (resid 15 and name ca resid 15 and name c ) 1 0 60 0 50 0 2 'HN(C0)H3 assign (resid 18 and name c resid 19 and name n ; _ (resid 19 ana name ca resid 19 and name c ) 1 0 -120 0 50 0 2 ' KNHA 8.9 Hz assign (resid 27 and name c resid 28 and name n ) (resid 28 and name ca resiα 28 and name c ) 1 0 -120 0 ~> 0 0 2 1 KNHA 7.6 Hz assign (resid 29 and name c resid 30 and name n ) (resid 30 and name ca resid 30 and name c ) -73 0 50 0 2 assign (resid 31 and name c resid 32 and name n ) (resiα 32 and name ca resid 32 and name c ) 1 3 -34 0 50 0 2 assign (resid 32 and name c resid 33 and name n ' (resid 33 and name ca resid 33 and name c , 1 3 60 0 50 0 2 'HN(CO)HE assign (resid 33 and name resid 34 and name n ' iresid 34 and name resid 34 and name c ' 1 -120 0 50 0 2 'KNHA 8.2 Hz assign (resiα 34 and name resid 35 and name n (resiα 35 and name resid 35 and name c 1 3 -120 0 5C 0 2 'KNHA 8.45 H assign resιd 36 and name resid 37 ar.α name r. (resid 37 and name ca resid 37. and name c ) 1 c -120 0 50 0 'HNHA 8.6 HZ assign (resid 38 and name c resid 39 ana name n (resid 39 and name ca resid 39 and name c 1 1 n -120 0 50 0 2 ΗNHA 8.6 Hz assign ( resiα 39 and name c resiα 40 ana name n l resid 40 and name ca resid 40 ana name c 1 " -120 0 50 0 2 'HNHA 8.3 hz assign resid 4 J and name c ) resiα 1 ana name n / resid and name ca) r siα 41 ana name c ) i .0 -120 1..0 50.0 2 'HNHA 8.3 Hz assign 'resid 41 and name c / resiα 42 ana name n 1 resid 42 and name ca) (resid 42 ana name c .0 -120 1..0 50.0 2 'HNHA 8.5 nz assign resiα 49 ana name c ; resiα 5C ana name n ( resid 5C and name ca) ' resiα 50 ana name c τ_ 0 -75 >..0 50.0 2 assign (resid 50 ana name c ) resiα 51 ana name n , resiα 51 and name ca) resid 51 ana name c 0 -120 1..0 53.0 2 ' HNHA 3.5 .-iz assign ' resid ... and name c ) resid 53 ana name n 'resiα 53 and name ca) resiα 53 and name c 1 0 60. .0 50.0 2 'HN(C0)H3 assign resid 55 ana name c ) ( esid 56 ana name n (resid 56 and name ca) i resiα 56 and name c 0 -60. .0 50.0 2 'HNHA 5.0 Hz assign (resid 58 and name c ) i resid 59 and name n (resid 59 and name ca) ( esid 59 ana name c 1 0 -120. .0 50.0 2 'HNHA 8.8 Hz assign (resid 59 and name c ) (resid 60 and name n (resid 60 ana name ca) (resiα 60 and name c 1 0 -120. .0 50.0 2 'HNHA 3.2 Hz assign (resid 60 and name c ) (resid 61 and name n (resid 61 and name ca) ( esid 61 and name c 1 0 -120. .0 50.0 2 'HNHA 8.1 Hz assign (resid 61 and name c ) (resid 62 and name n (resid 62 and name ca) (resid 62 and name c 1 0 -150. .0 70.0 2 !HNHA 7.9 Hz assign (resid 62 and name c ) (resid 63 and name n (resid 63 and name ca) (resid 63 and name c 1 0 -120. .0 50.0 2 'HNHA 8.7 Hz assign (resid 73 and name c ) (resid 74 and name n v resid 7 and name ca) ( resid 74 and name c _ 0 -120. .0 50.0 2 assign ( resiα 74 ana name c ) iresid -"5 and name n (resid 75 and name ca) (resid 75 and name c 1 0 -120. .0 50.0 2 'HNHA 9.15 Hz assign (resid 76 and name C 1 ( esid 77 and name n (resid 77 and name ca) (resid 77 and name c ) 1 0 -120. .0 50.0 2 'HNHA 3.2 Hz psi restraints assign (resid 15 and name n (resid 15 and name ca (resid 15 and name c 'resid 16 and name n ) 1 .0 50 . " • 50 .0 assign (rεsid 19 and name n ) (resid 19 and name ca (resid 19 and name c ) (resid 20 and name n ) 1 .0 120 .0 75 .0 2 assign (resid 25 and name n (resid 25 and name ca resid 25 and name c ) (resid 26 and name n 1 .0 120 .0 ^5 .0 2 assign resid 27 and name n ) (resid 27 and name ca resid 27 and name c ) (resid 28 and name n 1 .0 120 0 π 5 .0 2 assign resid 23 and name n ) (resiα 28 and name ca resid 23 and name c ) (resiα 29 and name n 1 .0 120 0 75 0 2 assign rεsid 29 ana name n ) (resid 29 and name ca rεsiα 29 and name c 1 ( resiα 30 ana namε n 1 .0 120 60 0 2 assign resiα 30 ana namε n (resid 30 and name ca resid 30 and name c ) 'resid 31 ana name n 1 .0 125 0 75 0 2 assign resiα 31 and name n ) (resid 31 and name ca resid 31 and name c ) (resid 32 and name n 1 0 120 0 75 0 2 assign resid 32 and name n ) (resid 32 and name ca resiα 32 and name c ) (resid 33 and name n 1 0 174 0 50 0 2 assign resid 33 and name n ) (resid 33 and name ca resid 33 and namε c ) (resid 34 and namε n 1 0 60 0 50 0 2 assign resiα 34 and name n ) (resiα 34 and name ca resid 34 and name c ) iresid 35 and name n 1 0 159 c 50 0 2 assign resid 35 and name n ) (resid 35 and name ca) resid 35 and name c ) (resid 36 and name n ) 1 0 156 0 50 0 2 assign. resid 36 and name n ) (resid 36 and name ca) resid 36 and name c ) (resid 37 and name n ) _ 120. 0 60 0 2 assign resid 37 and name n. ) iresiα 3 """ and name ca) resiα 37 ana name c ) (resid 38 ana name n ) 1 0 120. Q 60. 0 2 assign ( resid 40 ana name n / resid 40 ana name ca) resiα 40 ana name c ) iresid 41 ana name n ) 1 0 120. 0 60. 0 2 assign ( rεsiα 41 and name n ) (resid 41 ana name ca) rεsid 41 and name c ) i resiα 42 ana name n ) 1 0 120. 60. assign. ( rεsid 42 ana name n ) (resi 42 and name ca) resid 42 and name c ) (resid 43 and name n ) τ_ 0 120. 0 60. 0 2 assign. ' resid 43 ana name n ) ' resid 43 ana name ca) rεsiα 43 and na e c * 'resiα 44 ana namε n ) 1 2 120. 2 60. 0 assign ( resic 45 ana name n ; iresiα and name ca ) (resiα 45 a a name C ; iresiα 46 ana name n j τ_ 0 -50.0 50.0 2 HNHB ass gn. (resiα 51 and name n ) iresiα 31 ana name ca iresiα 51 and name c ( resid 52 and- name n ) _ 0 120.0 75.0 2 assign. (resiα 53 ana name n ! ' resiα 53 and name ca iresid 53 and namε c * ! resiα 5- a a name n 0 50.0 50. C 2 assign. resiα 58 ana name n ) l resiα 53 and name ca resid 53 ana name c (resiα 59 ana name n. 1 1 0 -60. 50. C 2 -iMriB assi n. resiα 59 ana namε n ; rεsiα 39 ana name ca resid 59 ana name c ) ( rεsiα 60 and name n 1_ 0 33.0 50.0 2 assign resid 60 ana name n ; "esi 60 ana name ca rεsid 60 ana name c ) (rεsiα 61 and name n 0 162.0 50.0 2 assign rεsid 61 ana name n ) ( rεsid 61 and name ca resid 61 and name c ) ( resiα 62 and name n 1 0 120.0 60.0 2 assign resid 62 and name n ) (resid 62 and name ca resiα 62 ana name c ) (resiα 63 ana name n 1 0 120.0 60. C 2 assign resid 63 and name n ) (rεsid 63 and name ca resid 63 and name c ) (resiα 64 and name n 1 0 165.0 50.0 2 assign resid 74 and name n ) (resid 74 and name ca resid 74 and name c ) (resid 75 and name n 1 0 120.0 60. C 2 assign resid 75 and name n ) (resid 75 and namε ca resid 75 and name c ) (resiα 76 ana name n 1 0 120.0 60.0 2 assign resid 76 ana name n ) (resiα " " 6 and name ca resid 76 and name c ) (rεsiα 77 ana name n \ 0 120.0 60.0 2 assign resid 73 and name n ) (rεsid 78 and name ca resid 78 and name c ) (rεsid 79 and name n I 0 120.0 60.0 2 assign resid 83 and name n ) (resid 83 and name ca resiα 83 and name c ) (resid 84 ana name n 1 0 120.0 60.0 2 assiαn resid 92 and name "" ) (resid 92 and name ca ( resid 92 and name c ) (rεsid 93 ana name n 1 0 120.0 75.0 2 1 chι-2 restraints assign (resid 2 and name ca) (resid 2 and name cb) (resid 2 and name cgl) (resid 2 and name edl) 1 1..00 -60 0 40 0 2 !LRCH (resid 31 and name ca) (resiα 31 and namε cb) (rεsid 31 and name eg) (resiα 31 and name edl) 1.0 180 0 40 0 2 'LRCH assign ( r;esid 32 and name ca) v resια 32 and name CD) resid 32 ana name eg) (rεsid 22 ana name edl) 1.0 180 0 40 0 ' RCH assiαn resiα 74 and name ca) ' resiα 74 ana name CD) (resiα 74 and name cgl) (resiα " ana name cαl 1.0 -60 0 40 2 ' LRCH assiαn (resid 90 and name ca; (resiα 90 a a name cb; (resiα 90 ana name cgl) (resid 90 ana name edl) 1.0 180 0 40 'LRCH Example 3: Identification of blocking antibodies using a competitive binding assay and immobilised wild type GST-MSP-1 19 . In previous studies antibodies that blocked the action of the neutralising antibodies 1 .8 and 12.10 had been defined either directly in the MSP-1 2 processing assay (Blackman et al. 1994) in a coupled erythrocyte invasion-MSP-l 2 processing assay (Guevara et al, 1997) or in a competitive radioimmunoassay with merozoite protein as the antigen (Guevara et al, 1997). These studies have been extended using recombinant MSP-1 and BIAcore analysis. A recombinant fusion protein comprising wild type MSP-11 9 fused to GST was coupled to the sensor chip and competitor antibody was first allowed to bind to the antigen. Then a solution of either mAb 12.8 or 12.10 was passed over the chip and the amount of binding of this second antibody was quantified. If the first antibody interferes with the binding of the second antibody then this is reflected in a reduction in the amount of second antibody bound. Methods The wild type GST-MSP-1 ! was coupled to a CM5 sensor chip. The binding assays were performed with a constant flow rate of 5 μl min "1 at 25°C. For binding, purified mAbs 1E1, 8A12 and 2F10 at 100 μg ml "1 in HBS-EP buffer (lOmM HEPES pH7.4 containing 150mM NaCl, 3mM EDTA and 0.005%v/v polysorbate 20); mAbs 1E8, 9C8, 12D11, 111.2 and 11 1.4 in cell culture medium supernatant; mAbs 2.2, 7.5 and 89.1 at 1 :10 dilution of ascitic fluid in HBS-EP buffer; and mouse α-GST antibody at 1 : 10 -dilution serum in HBS-EP buffer were allowed to interact with immobilised wild type GST-MSP- l ι 9 for 10 min. After allowing 5 min for dissociation of low affinity interactions, either mAb 12.8 or 12.10 at 100 μg ml "1 in HBS-EP buffer was added and allowed to bind for 10 min. After washing the chip for 5 min the binding of 12.8 or 12.10 was measured. The chip was regenerated by washing off bound antibody with 10 mM glycine-HCl, pH 2.4. or when required with 100 mM glycine-HCl, pH 1.8, for 3 min. Results The results are shown in Figure 12. All the competitor antibodies bind to the GST-MSP- 1 19 antigen, with the exception of mAb89.1 which is a negative control. As expected, mAbs 12.8 and 12.10 competed with each other (Guevara et al, 1997). The other antibodies which do not inhibit processing could to greater or lesser interfere with the binding of 12.8 and 12.10. As expected from previous studies mAbs 1E1, and 7.5 blocked both 12.8 and 12.10, whereas 2.2 and 111.4 blocked 12.8. Another particularly effective blocking antibody identified in this study was mAb9C8. Example 4: Immunization of small animals with modified GST-MSP-1 and anaivsis of the antibodies induced To determine whether or not the modified proteins were immunogenic, recombinant GST- MSP- 1 19 fusion proteins were used to raise antibodies by immunisation. Methods Two modified proteins containing either 3 [27+31+43] or 4 [15+27+31+43] amino acid substitutions, respectively, were used to immunise rabbits and mice. The rabbits were immunised subcutaneously with MSP-1 19 protein in Freund's complete adjuvant and then boosted on three occasions with 200 μg of the protein in Freund's incomplete adjuvant 21, 42 and 63 days later, and serum samples were collected. The presence and level of antibodies binding to the native MSP-1 protein in the parasite was assessed by indirect immuno fluorescence using acetone fixed smears of parasite- infected erythrocytes. The sera were diluted serially in phosphate buffered saline (PBS) and incubated on the slide for 30 min at room temperature. After washing, the slides were incubated with FITC conjugated goat anti-rabbit or anti-mouse IgG, washed, and then examined by fluorescence microscopy. The sera were also analysed in an MSP-1 secondary processing assay. Analysis and quantitation of secondary processing of MSP-1 in merozoite preparations was by a modification of an assay described previously (Blackman et al, 1994). Washed P. falciparum 3D7 merozoites were resuspended " in ice-cold 50 mM Tris-HCl pH 7.5 containing 10 mM CaC and 2 mM MgCl 2 (reaction buffer). Aliquots of about 1 x IO 9 merozoites were dispensed into 1.5 ml centrifuge tubes on ice. and the parasites pelleted in a microfuge at 13.000 x g for 2 minutes at 4°C. The supernatant was removed, and individual merozoite pellets were then resuspended on ice in 25 μl of reaction buffer further supplemented with protease inhibitor or antibodies as appropriate. Merozoites were maintained on ice for 20 min to allow antibody binding, then transferred to a 37°C water bath for one hour to allow processing to proceed. Assays always included the following controls: a "positive processing" control sample of merozoites resuspended in reaction buffer only; a "negative processing" control sample of merozoites resuspended in reaction buffer plus ImM PMSF; and a zero time (Oh) control, in which processing was stopped before the 37°C incubation step. The processing was assayed using the western blot-based method and by a modified processing assay. Supematants from the assays were obtained after centrifugation for 30 min at 4°C, 13.000 x g to remove the insoluble material. The amount of MSP-1 33 in the supematants was measured using an ELISA method. Fifty microlitres of diluted sample supematants were added to the wells of an ELISA plate (NUNC F96 Cert. Maxisorp) that had been coated with 100 μl/well of 4 μg ml "1 human mAb X509 in PBS. Plates were incubated for 4 hours at 37°C and then washed 3 times with 0.01% PBS-Tween (PBS-T). Bound MSP-1 33 protein was detected by addition of 100 μl of 1 :4000 dilution of mouse mAb G13 for 1 hour at 37°C, followed by washing and the addition of 100 μl of 1:1000 dilution of sheep anti-mouse IgG (H+L) HRP-conjugated antibody. After incubation for 1 h at 37°C, the plates were washed again and HRP was detected by the addition of 100 μl of freshly prepared substrate solution (400mg l "1 o-phenylenediamine dihydrochloride in 0.05 M phosphate buffer, 0.024 M citric acid and 0.012% H 2 0 2 ) at room temperature for 20 min. The reaction was stopped by adding 10 μl of 1 M sulphuric acid and the absorbance of each sample was measured at 492 nm. Results The results are shown in Figure 13. The two modified proteins produced antibodies that reacted with MSP-1 in the parasite-infected erythrocyte, with a serum titre of 1 :10.000, which was an identical titre to that of a serum produced in the same way by immunisation with a recombinant protein containing the wildtype MSP-1 sequence. This indicates that the modified proteins can produce antibodies that react with the native protein. The antibodies induced by immunization were able to partially inhibit processing at the concentration used in a preliminary experiment, whereas in the control serum no antibodies that inhibited processing were present. Example 5: Design and Synthesis of a Plasmodium Falciparum Merozoite Surface Protein-1 Gene Fragment Optimized for Pichia Pastoris Heterologous Expression The coding sequence of the Plasmodium falciparum merozoite surface protein-1 (MSP1) 41.1 kDa processed fragment (MSP-142) has been redesigned for optimal heterologous expression in the yeast Pichia pastoris. The optimized DNA sequence was synthesized by PCR gene assembly, in the form of two fragments, MSP-133 and MSP-119. P. pastoris was transformed with an expression vector containing the optimized MSP-119 construct. Recombinant strains were shown to express high levels of non-glycosylated, properly folded MSP-119 protein. Proteins encoded by the AT-rich genome of the human malaria parasite Plasmodium falciparum are generally poorly expressed in heterologous systems (Withers-Martinez et al., 1999). The methylotrophic yeast Pichia (Komagataella) pastoris is an appropriate system for expression of disulphide-bridged proteins such as the C-terminal fragment of the P. falciparum merozoite surface protein-1 (MSP1) (White et al., 1994; Morgan et al., 1999). In the P. pastoris expression system, it is important to avoid premature transcription termination due to AT-rich stretches (Romanos et al., 1991). Codon preferences for highly expressed genes in P. pastoris have been identified (Sreekrishna et al., EP 0 586 892 Al). Therefore, a synthetic MSP-142 gene fragment with codon usage optimized for P. pastoris expression was designed, using novel computer software (Withers-Martinez et al., 1999). It has previously been shown that the MSP-119 fragment is partially glycosylated when expressed in P. pastoris, and the carbohydrate must be e zymatically removed during purification (Morgan et al., 1999). Therefore, two specific point mutations were introduced into the synthetic MSP-142 protein sequence in order to prevent N-linked glycosylation at NxS/T sites (one potential site within the MSP-133 sequence, and a known site within the MSP-1 19 sequence at Asn 1). The optimized MSP-142 sequence was synthesized by gene assembly polymerase chain reaction (Stemmer et al.. 1995. Withers-Martinez et al.. 1999), in the form of separate MSP-133 and MSP-119 fragments. The -optimized MSP-1 19 fragment was subcloned into a novel modified Pichia expression vector, transformed into the P. pastoris host strain SMD1168. and several independent transformants were isolated. There transformants were shown to efficiently express non-glycosylated, properly folded MSP-119. Strong expression of the optimized gene was observed in low copy number transformants. A multiple copy transformant with intermediate level G418 resistance gave expression of purified MSP-119 at a level equal to the high-expressing strain previously described (Morgan et al., 1999), which contains the original P. falciparum DNA. Thus, it should be possible to obtain even higher yields from high level G418-resistant transformants of the synthetic optimized gene. Methods: Gene Assembly The P. falciparum MSP-142 (41.1 kDa) fragment protein sequence SWISS-PROT accession number P04933: positions 1264-1621) was first altered to eliminate N-linked glycosylation signals by 2 amino acid substitutions. The sequences NYT (in the N- terminal portion; position 1445) and NIS (at the beginning of the C-terminal fragment; position 1526) were changed to QYT and NIA respectively. The protein sequence was then reverse-translated with DNA-STAR using the S. cerevisiae codon preferences. This sequence was used as input for the CODOP program (Withers-Martinez et al., 1999). Ten random sequences were generated with this program, using a codon weighting table (Figure 14) derived from codon usage in highly expressed P. pastoris genes (Sreekrishna et al.. EP 0 586 892 Al). Thus, the codon table should reflect usage in highly expressed genes, rather than average usage. The random sequence that contained the minimum number of unfavourable codons (6) was selected, and these codons were changed manually to more preferred alternatives- The sequence was then analysed with DNA- STAR to check for AT-rich sequences that may cause transcription termination, and for direct and inverted repeats. A set of 50 overlapping oligonucleotides coding for the final sequence was then generated. This consisted of 49 oligonucleotides of length 42 nt, and one of length 48 nt. Each oligonucleotide had a 21 bp overlap with its neighbours, with no gaps. Estimated T m s were in the range of 60°C to 77°C. Oligonucleotides were synthesised by Oswel (Southampton, UK) at 40 nmol scale, and supplied in deionised water without purification. Outside primers of various lengths for the amplification step were also synthesised, to give a T m of 62°C to 64°C, and contained a 5 '-terminal phosphate group for ligation following the amplification step. The reverse primers also included a translation termination codon (UAA in the complementary strand). All oligonucleotides were diluted to 10 μM in ddH 2 0 before use. The PCR-mediated gene assembly and amplification were carried out as described (Stemmer et α/., 1995; Withers-Martinez et al., 1999), using a Biometra cycler, in thin- walled 200 μL tubes, under the following conditions. Gene assembly reactions (Reaction 1): 50 μL volume 2 units Vent polymerase (New England Biolabs) 0.4 mM dNTPs 1 x Vent polymerase buffer Oligonucleotide mix containing each oligonucleotide at 200 nM Cycles: 32 cycles (2 h 33 m) denaturation 94°C 30 s annealing 52°C 30 s extension 72°C 3 m Three fragments of the MSP-142 (41.1 kDa) region were synthesised separately with different outside primers and subsets of the 50 oligonucleotide set: N-terminal fragment (bp 1-423) 21 oligonucleotides middle fragment (bp 337-786) 22 oligonucleotides C-terminal fragment (bp 787-1074) 14 oligonucleotides The C-terminal fragment produces a 10.6 kDa fragment (MSP-1 19). The N-terminal and middle fragments, which overlap between positions 337 and 423, were subsequently spliced together at the Bglll site (371-376) to give a 786 bp fragment that encodes the 30.5 kDa MSP- 133 protein. Amplification reactions (Reaction 2): 100 μL volume 10 μL aliquot of the gene assembly reaction 4 units Vent polymerase 0.4 mM dNTPs 1 x Vent polymerase buffer 1 μM outside primers Cycles: 32 cycles (2 h 55 m) denaturation 94°C 45 s annealing 52°C 45 s extension 72°C 3 m final extension 72°C 5 m The PCR products were then purified by filtration with Centricon-100 units (Amicon), and cloned directly into the vectors by blunt-end ligation overnight at 16°C with T4 DNA ligase. The synthetic MSP-1 19 gene was cloned directly into a P. pastoris expression vector. The modified pPIC9KHXa vector, containing a His 6 tag and factor Xa cleavage site (see Figure 15) inserted in the pPIC9K SnaBI site, had been digested with Pmll and treated with calf alkaline phosphatase. The HXa vector had been previously created by insertion of a 36 bp synthetic oligonucleotide. containing the His 6 tag. factor Xa cleavage site, and Pmll restriction site into the SnaBI site of the pPIC9K vector. The N-terminal and middle fragment PCR products were cloned into the Smal site of the dephosphorylated pUCl lδ vector. Plasmid clones containing inserts were sequenced. Clones with the correct synthetic sequence were then digested and the two fragments were gel-purified. The N-terminal fragment clones were digested with EcoRI and Bglll, and the middle fragment clones were digested with Hindlll and Bglll. The recombinant fragments were purified on an agarose gel, and eluted with a QIAGEN extraction kit. The purified N-terminal and middle fragments were then spliced together by ligation into a pUC118 vector that had been digested with Hindlll and EcoRI and treated with calf alkaline phosphatase. This created the complete synthetic MSP-133 coding sequence. The N- terminal and middle fragment PCR products were cloned into the Smal site of the dephosphorylated pUC118 vector. Plasmid clones containing inserts were sequenced. Clones with the correct synthetic sequence were then digested and the two fragments were gel-purified. The N-terminal fragment clones were digested with EcoRI and Bglll, and the middle fragment clones were digested with Hindlll and Bglll. The recombinant fragments were purified on an agarose gel, and eluted with a QIAGEN extraction kit. The purified N-terminal and middle fragments were then spliced together by ligation into a pUCl lδ vector that had been digested with Hindlll and EcoRI and treated with calf alkaline phosphatase. This created the complete synthetic MSP-133 coding sequence. Methods: Expression and Purification The methylotrophic yeast Pichia (Komagataella) pastoris strain SMD1168 was transformed by electroporation as described previously (Morgan et al., 1999). In addition, some G418-resistant clones were isolated using Hybond-N+ membranes (Fairlie et al. 1999). Expression screening of transformants -was performed by growing 10 ml cultures in buffered minimal glucose medium. Cells were harvested and resuspended in 10 ml buffered minimal methanol medium at 1.0 OD 6 oo and grown overnight to a final ODβoo of 2.5 to 3.0. Cells were removed by centrifugation-.- and 1.2 ml of the supernatant medium was precipitated 30 min on ice with 15 % trichloroacetic acid. The samples were centrifuged for 30 min at 14000 rpm at 4 °C in a micro fuge, and the protein pellets were washed twice with cold acetone. Samples were resuspended in 12 μl ddH 0, and 5 μl was electrophoresed, after reduction with DTT, on NOVEX pre-poured acrylamide gels according to manufacturer's instructions. NOVEX 4-12 % acrylamide gradient, or 10 % acrylamide, Bis/Tris gels in MES buffer were used. Protein gels were stained with Coomassie colloidal Brilliant Blue stain (Sigma). Homogeneously purified MSP-1 19 was obtained as described previously (Morgan et al., 1999), except that enzymatic deglycosylation was omitted for the synthetic gene products. Methods: NMR One-dimensional Η- and 2-dimensional {Η/ N}-HSQC spectra were acquired as described previously (Morgan et al., 1999), at 25 °C, at sample concentrations of 1.1-2.5 mM. Results The sequences of the synthetic DNA fragments, and the resulting predicted protein products, are shown in Figure 15. A summary of the resulting improvements to the sequence is given in Table 3. Total codons P. pastoris preferred Unfavourable % AT content codons codons P. falciparum MSP1 358 140 28 74 41.1 kDa fragment Synthetic 41.1 kDa 358 276 58 fragment Table 3. Codon usage PCR-gene assembly reactions for the MSP-133 (two sections) and MSP-119 synthetic fragments are shown on agarose gels in Figure 16. This demonstrated that a single, correct size major product was observed in each case. The PCR products were subcloned, screened, and sequenced as described in the Methods section. P. pastoris was transformed with the synthetic MSP-1 19 construct in the modified pPIC9K expression vector (pPIC9K-HXa: Figure 15). Expression of the synthetic MSP- 119 product in three independent transformants is shown on a protein gel in Figure 17. The protein samples were prepared by trichloroacetic acid precipitation from culture supematants as described in the Methods section. This demonstrated that a single, major product was present in each sample, corresponding to the expected migration of the synthetic MSP-119 protein. This migrated slightly more slowly than the control .sample, which as described previously (Morgan et al., 1999) has a shorter N-terminal tag sequence. There was no trace of heterogeneous, slowly migrating recombinant protein that would result from glycosylation. Therefore, non-glycosylated, synthetic MSP-119 is efficiently expressed by the transformed yeast. The yield (measured by UV absorbance) of purified MSP-119 was 16 mg/L for low copy number transformants (resistant to 0.25 mg/ml G418), and increased to 24 mg/L for intermediate G418 resistance (resistant to 1.0 mg/ml G418). This can be compared -with yields of 1-2 mg/L for low copy number transformants of P. pastoris with the original Plasmodium falciparum coding sequence. before isolation of a highly G418-resistant strain (Morgan et al., 1999). This indicated that the synthetic MSP-1 19 construct is advantageous for recombinant protein expression, and that fuπher improvement would result from isolation of higher copy number transformants. -3 One-dimensional proton NMR experiments demonstrated that the synthetic MSP-119 protein spectrum was very similar to the previously studied protein (Morgan et al., 1999), and represented a correctly folded protein (data not shown). This was further confirmed by a 2D-{ 'H/ 1; N}-HSQC spectra (Figure 18), which also shows that the structure of the 0 synthetic product is identical to the previously studied protein, except for slight differences at the N-terminus which are consistent with the presence of a distinct N- terminal tag sequence, and S3->A mutation at the glycosylation site. Backbone NH proton and l3 N chemical shifts for the original P. falciparum sequence product have been previously presented (Morgan et al., 1999). The similarity between the two spectra, outside of the N-terminal region, is strong evidence that both protein forms are in a structurally similar, correctly folded state. References Abseher. R.. Horstink. L.. Hilbers. C. W. & Nilges. M. (1998). Essential spaces defined by NMR structure ensembles and molecular dynamics simulation show significant overlap. Proteins: Structure, Function and Genetics, 31, 370-382. Barbato, G., Ikura, M., Kay, L. E., Pastor, R. W. & Bax, A. (1992). 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Claims: CLAIMS 1. A non-naturally occurring variant of a C-terminal fragment of a Plasmodium merozoite surface protein-1 (MSP-1) wherein said variant has (i) a reduced affinity, compared with a naturally occurring Plasmodium MSP-11 9 , for at least one first antibody capable of blocking the binding of a second antibody, which second antibody inhibits the proteolytic cleavage of Plasmodium MSP-1 2 and (ii) substantially the same affinity for at least one third antibody compared with said naturally occurring Plasmodium MSP-1 19 . which third antibody inhibits the proteolytic cleavage of Plasmodium MSP-1 42 . 2. A variant according to claim 1 wherein said Plasmodium MSP-1 19 and MSP-1 42 are Plasmodium falciparum MSP-11 9 and MSP-1 42 . 3. A variant according to claim 1 or 2 wherein the first antibody is selected from mAbs 1E1, 2.2, 7.5, 9C8 and 111.4. 4. A variant according to claim 1 or 2 wherein the second and/or third antibody is selected from mAbs 12.8, 12.10 and 5B1. 5. A non-naturally occurring variant of a C-terminal fragment of a Plasmodium merozoite surface protein-1 (MSP-1) comprising an amino acid modification at any one of amino acid residues 14, 15, 27, 31, 34 , 43, 48 and 53 of the Plasmodium falciparum MSP-1 19 amino acid sequence shown as SEQ I.D. No. 1 or their equivalent positions in other Plasmodium MSP-1 1 polypeptides. 6. A variant according to claim 5 wherein said modifications are selected from Glnl4→Arg, Glnl4→Gly, AsnlS→Arg, Glu27→Tyr, Leu31→Arg, Tyr34→Ser, Tyr34→Ile, Glu43-»Leu, Thr48-»Lys and Asn53→Arg and their equivalents in other Plasmodium MSP-1 19 polypeptides. 7. A variant according to claim 6 wherein said substitutions are selected from [Glu27→Tyr, Leu31→Arg and Glu43→Leu], [Glu27→Tyr, Leu31→Arg, Tyr34→Ser and Glu43→Leu], [AsnlS→Arg, Glu27→Tyr, Leu31-»Arg and Glu43—>Leu] and their equivalents in other Plasmodium MSP-1 19 polypeptides. 8. A variant according to any one of claims 5 to 7 further comprising a mutation at Cysl2 and/or Cys28 of the Plasmodium falciparum MSP-1 19 amino acid sequence shown as SEQ I.D. No. 1. 9. A variant according to claim 8, in which the mutation is a substitution selected from Cysl2-→Ile and Cys28-→Trp, and Cysl2→Ala and Cys28→Phe. 10. A variant according to claim 9, in which the substitutions are selected from [Cysl2→Ile, Asn 15-* Arg, Glu27→Tyr, Cys28-→Trp, Leu31→Arg, Glu43→Leu], [Cysl2→Ile, Asn 15-→Arg, Glu27→Tyr, Cys28→Trp, Leu31-→Arg, Glu43→Leu, Asn53→Arg], [Cysl2→Ile, Asn 15→Arg, Glu27→Tyr, Cys28→Trp, Leu31-→Arg, Tyr34→Ser. Glu43→Leu, Asn53→Arg] and their equivalents in other PlasmodiumMS?- 119 polypeptides. 11. A method for producing a Plasmodium MSP-1 variant for use in preparing a vaccine composition which method comprises modifying one or more amino acid residues of a Plasmodium MSP-1 C-terminal fragment such that the resulting derivative has (i) a reduced affinity, compared with a naturally occurring Plasmodium MSP-1 19 , for at least one first antibody capable of blocking the binding of a second antibody, which second antibody inhibits the proteolytic cleavage of Plasmodium MSP-1 42 and (ii) substantially the same affinity for at least one third antibody compared with said naturally occurring Plasmodium MSP-1 19 , which third antibody inhibits the proteolytic cleavage of Plasmodium MSP-1 42 . 12. A non-naturally occurring Plasmodium MSP-1 variant obtained by the method of claim 11. 13. A polynucleotide encoding a variant according to any one of claims 1 to 10 or 12 operably linked to a regulatory sequence capable of directing the expression of said nucleotide in a host cell. 14. A polynucleotide according to claim 13 having a sequence optimised for expression in said host cell. 15. A polynucleotide according to claim 13 or 14. in which the host cell is a Pichia pastoris cell. 16. A nucleic acid vector comprising a polynucleotide according to claim 13, 14 or 15. 17. A host cell comprising a vector according to claim 16. 18. A pharmaceutical composition comprising a variant according to any one of claims 1 to 10 or 12, a polynucleotide according to any one of claims 13 to 15 or a vector according to claim 16 together with a pharmaceutically acceptable carrier or diluent. 19. A composition according to claim 18 further comprising an immunogenic Plasmodium polypeptide or fragment or derivative thereof. 20. A method for producing anti-MSP-1 antibodies which method comprises administering a polypeptide according to any one of claims 1 to 10 or 12, a polynucleotide according to any one of claims 13 to 15 or a vector according to claim 16 to a mammal. 21. A method for producing polyclonal anti-MSP-1 antibodies which method comprises administering a polypeptide according to any one of claims 1 to 10 or 12, a polynucleotide according to any one of claims 13 to 15 or a vector according to cjaim 16 to a mammal and extracting the serum from said mammal. 22. An antibody produced by the method of claim 20 or 21. 23. A method of inducing immunity against malaria induced by Plasmodium falciparum which comprises administering to a person in need of such immunity an effective amount of the variant of claim 1, the polynucleotide of claim 13 or the vector of claim 16. 24. A method of immunizing a mammal, said method comprising administering an effective amount the polypeptide of claim 1. the polynucleotide of claim 13 or the vector of claim 16. 25. The method of claim 24, wherein said mammal is immunized against malaria. 26. A method of treating a malaria infection in a human patient which comprises administering to the patient an effective amount of the pharmaceutical composition of claim 18 or 19. 27. A variant according to any one of claims 1 to 10 or 12, a polynucleotide according to any one of claims 13 to 15 or a vector according to claim 16 for use in therapy. 24. A computer readable medium having stored thereon a model of the MSP-1 1 NMR stmcture. 25. A nucleic acid encoding a Plasmodium MSP-1 polypeptide, in which the nucleic acid is optimised for expression in a heterologous host cell. 26. A nucleic acid according to Claim 25, in which the heterologous host is a Pischia pastoris cell. 27. A nucleic acid according to Claim 25 or 26, in which the polypeptide is selected from an MSP-1 42 polypeptide comprising a sequence shown in Figures 2C and 2E, an MSP-11 9 polypeptide comprising a sequence shown in Figure 2C, and an MSP-1 33 polypeptide comprising a sequence shown in Figure 2E. 28. A nucleic acid according to any of Claims 25 to 27, in which the nucleic acid comprises a sequence selected from the sequences of Figure 2A, Figure 2B and Figure 2D. 29. A nucleic acid vector comprising a nucleic acid according to any of Claims 25 to 28. 30. A host cell comprising a vector according to claim 29. 31. A pharmaceutical composition comprising a nucleic acid according to any of Claims 25 to 27 or a vector according to Claim 29 together with a pharmaceutically acceptable carrier or diluent. 32. A composition according to Claim 31 further comprising an immunogenic Plasmodium polypeptide or fragment or derivative thereof.

Inventors: BIRDSALL BERRY, FEENEY JAMES, HOLDER ANTHONY, MORGAN WILLIAM, SYED SHABIH, UTHAIPIBULL CHAIRAT, BIRDSALL, BERRY, FEENEY, JAMES, HOLDER, ANTHONY, MORGAN, WILLIAM, SYED, SHABIH, UTHAIPIBULL, CHAIRAT
Applicants: BIRDSALL BERRY, FEENEY JAMES, HOLDER ANTHONY, MEDICAL RES COUNCIL, MORGAN WILLIAM, SYED SHABIH, UTHAIPIBULL CHAIRAT, BIRDSALL, BERRY, FEENEY, JAMES, HOLDER, ANTHONY, MEDICAL RESEARCH COUNCIL, MORGAN, WILLIAM, SYED, SHABIH, UTHAIPIBULL, CHAIRAT
Application year: 2000
Main classification: C07K14
IPC codes: A61K31, A61K38, A61K39, A61K48, A61P33, C07K14, C07K16, C12N1, C12N15, C12P21, C12R1