bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of protein transmembrane helical regions by a neural network
Authors: Dombi George W. , Lawrence Jeannette
Published: July 2010

Neural networks were used to generalize common themes found in transmembrane‐spanning protein helices. Various‐sized databases were used containing nonoverlapping sequences, each 25 amino acids long. Training consisted of sorting these sequences into 1 of 2 groups: transmembrane helical peptides or nontransmembrane peptides. Learning was measured using a test set 10% the size of the training set. As training set size increased from 214 sequences to 1,751 sequences, learning increased in a nonlinear manner from 75% to a high of 98%, then declined to a low of 87%. The final training database consisted of roughly equal numbers of transmembrane (928) and nontransmembrane (1,018) sequences. All transmembrane sequences were entered into the database with respect to their lipid membrane orientation: from inside the membrane to outside. Generalized transmembrane helix and nontransmembrane peptides were constructed from the maximally weighted connecting strengths of fully trained networks. Four generalized transmembrane helices were found to contain 9 consensus residues: a K‐R‐F triplet was found at the inside lipid interface, 2 isoleucine and 2 other phenylalanine residues were present in the helical body, and 2 tryptophan residues were found near the outside lipid interface. As a test of the training method, bacteriorhodopsin was examined to determine the position of its 7 transmembrane helices.
Models of the serine protease domain of the human antithrombotic plasma factor activated protein C and its zymogen
Authors: Fisher Cindy L. , Greengard Judith S. , Griffin John H.
Published: July 2010

Three‐dimensional structural analysis of physiologically important serine proteases is useful in identifying functional features relevant to the expression of their activities and specificities. The human serine protease anticoagulant protein C is currently the object of many genetic site‐directed mutagenesis studies. Analyzing relationships between its structure and function and between naturally occurring mutations and their corresponding clinical phenotypes would be greatly assisted by a 3‐dimensional structure of the enzyme. To this end, molecular models of the protease domain of protein C have been produced using computational techniques based on known crystal structures of homologous enzymes and on protein C functional information. The resultant models corresponding to different stages along the processing pathway of protein C were analyzed for structural and electrostatic differences arising during the process of protein C maturation and activation. The most satisfactory models included a calcium ion bound to residues homologous to those that ligate calcium in the trypsin structure. Inspection of the surface features of the models allowed identification of residues putatively involved in specific functional interactions. In particular, analysis of the electrostatic potential surface of the model delineated a positively charged region likely to represent a novel substrate recognition exosite. To assist with future mutational studies, binding of an octapeptide representing a protein C cleavage site of its substrate factor Va to the enzyme's active site region was modeled and analyzed.
Heparin binding domain peptides of antithrombin III: Analysis by isothermal titration calorimetry and circular dichroism spectroscopy
Authors: Tyler‐Cross Ruth , Harris Robert B. , Sobel Michael , Marques Dalila
Published: July 2010

The serine proteinase inhibitor antithrombin III (ATIII) is a key regulatory protein of intrinsic blood coagulation. ATIII attains its full biological activity only upon binding polysulfated oligosaccharides, such as heparin. A series of synthetic peptides have been prepared based on the proposed heparin binding regions of ATIII and their ability to bind heparin has been assessed by CD spectrometry, by isothermal titration calorimetry, and by the ability of the peptides to compete with ATIII for binding heparin in a factor Xa procoagulant enzyme assay. Peptide F123‐G148, which encompasses both the purported high‐affinity pentasaccharide binding region and an adjacent, C‐terminally directed segment of ATIII, was found to bind heparin with good affinity, but amino‐terminal truncations of this sequence, including L130‐G148 and K136‐G148 displayed attenuated heparin binding activities. In fact, K136‐G148 appears to encompass only a low‐affinity heparin binding site. In contrast, peptides based solely on the high‐affinity binding site (K121‐A134) displayed much higher affinities for heparin. By CD spectrometry, these high‐affinity peptides are chiefly random coil in nature, but low μM concentrations of heparin induce significant α‐helix conformation. K121‐A134 also effectively competes with ATIII for binding heparin. Thus, through the use of synthetic peptides that encompass part, if not all, of the heparin binding site(s) within ATIII, we have further elucidated the structure‐function relations of heparin‐ATIII interactions.
The three‐dimensional profile method using residue preference as a continuous function of residue environment
Authors: Zhang Kam Y.J. , Eisenberg David
Published: July 2010

In the 3‐dimensional profile method, the compatibility of an amino acid sequence for a given protein structure is scored as the sum of the preferences of the residues for their environments in the 3D structure. In the original method (Bowie JU, Lüthy R, Eisenberg D, 1991, Science 253:164‐170), residue environments were quantized into 18 discrete environmental classes. Here, amino acid residue preferences are expressed as a continuous function of environmental variables (residue area buried and fractional area buried by polar atoms). This continuous representation of residue preferences, expressed as a Fourier series, avoids the abrupt change of preference of residues in slightly different environments, as encountered in the original method with its 18 discrete environmental classes. When compared with the discrete 18‐class representation of residue environments, this continuous 3D profile is found to be more sensitive in identifying sequences that fold into the profiled structure but share with it little sequence identity. The continuous 3D profile is also less sensitive to errors in environmental variables than is the discrete 3D profile. The continuous 3D profile can also be used to detect wrong folds or incorrectly modeled segments in an otherwise correct structure, as could the discrete 3D profile (Lüthy R, Bowie JU, Eisenberg D, 1992, Nature 356:83‐85). Moreover, the progress of structure improvement during atomic refinement can also be monitored by examining the profile scores in a moving‐window scan. Finally, by defining a functional form for profile scores, we open the way to profile atomic refinement in which an atomic structure adjusts to produce residue environments more compatible with the protein side chains.
Similarity between pyridoxal/pyridoxamine phosphate‐dependent enzymes involved in dideoxy and deoxyaminosugar biosynthesis and other pyridoxal phosphate enzymes
Authors: Pascarella Stefano , Bossa Francesco
Published: July 2010

A multiple sequence alignment among aspartate aminotransferase, dialkylglycine decarboxylase, and serine hydroxymethyltransferase (DAS) was used for profile databank search. The DAS profile could detect similarities to other pyridoxal or pyridoxamine phosphate‐dependent enzymes, like several gene products involved in dideoxysugar and deoxyaminosugar synthesis. The alignment among DAS and such gene products shows the conservation of aspartate 222 and lysine 258, which, in aspartate aminotransferase, interacts with the N1 of the coenzyme pyridine ring and forms the internal Schiff base, respectively. The lysine is replaced by histidine in the pyridoxamine phosphate‐dependent gene products. The alignment indicates also that the region encompassing the coenzyme binding site is the most conserved.
A role for surface hydrophobicity in protein‐protein recognition
Authors: Young L. , Jernigan R.L. , Covell D.G.
Published: July 2010

The role of hydrophobicity as a determinant of protein‐protein interactions is examined. Surfaces of apo‐protein targets comprising 9 classes of enzymes, 7 antibody fragments, hirudin, growth hormone, and retinol‐binding protein, and their associated ligands with available X‐ray structures for their complexed forms, are scanned to determine clusters of surface‐accessible amino acids. Clusters of surface residues are ranked on the basis of the hydrophobicity of their constituent amino acids. The results indicate that the location of the co‐crystallized ligand is commonly found to correspond with one of the strongest hydrophobic clusters on the surface of the target molecule. In 25 of 38 cases, the correspondence is exact, with the position of the most hydrophobic cluster coinciding with more than one‐third of the surface buried by the bound ligand. The remaining 13 cases demonstrate this correspondence within the top 6 hydrophobic clusters. These results suggest that surface hydrophobicity can be used to identify regions of a protein's surface most likely to interact with a binding ligand. This fast and simple procedure may be useful for identifying small sets of well‐defined loci for possible ligand attachment.
A β‐turn in α‐amanitin is the most important structural feature for binding to RNA polymerase II and three monoclonal antibodies
Authors: Baumann Karlheinz , Zanotti Giancarlo , Faulstich Heinz
Published: July 2010

Four amatoxin‐binding proteins with KD values in the nanomolar range, 3 monoclonal antibodies and RNA polymerase II, were studied with respect to their affinities to 24 α‐amanitin derivatives with modified side chains. From KD values we estimated the amounts of binding energy that single side chains of the amatoxins contribute to complex formation. Ile6, previously identified by X‐ray analysis to be part of a β‐turn (Kostansek EC, Lipscomb WN, Yocum RR, Thiessen WE, 1978, Biochemistry 17: 3790‐3795) proved to be of outstanding importance in all complexes. Replacement of the isoleucine with alanine reduced the affinity to all binding proteins to < 1%, suggesting a strong hydrophobic interaction. A strong effect was also seen when Gly5 was replaced with alanine, suggesting that the absence of a side chain in proximity to the β‐turn is likewise important. In addition to the β‐turn, each of the proteins showed at least 2 other points of strong contact formed by hydrogen bonds. Donors are the indole NH of 6′‐hydroxy‐Trp4 and OH of hydroxy‐Pro2 and dihydroxy‐Ile3. All the antibodies, but not RNA polymerase II, recognized the indole nucleus of 6″hydroxy‐Trp4. The geometric arrangement of the 4 strongest contact points suggests that the amatoxin binding site is different in each of the 4 proteins, except for the 2 antibodies raised in the same animal. Here, most of the contact points were identical but differed in strength of interaction. The method of structural analysis presented in this study is useful for identifying contact sites in complexes of proteins with peptides of rigid conformation. Furthermore, the method complements X‐ray data by providing information on the amount of binding energy contributed by single structural elements.
Structure of glutathione reductase from escherichia coli at 1.86 Å resolution: Comparison with the enzyme from human erythrocytes
Authors: Mittl Peer R.E. , Schulz Georg E.
Published: July 2010

The crystal structure of the dimeric flavoenzyme glutathione reductase from Escherichia coli was determined and refined to an R‐factor of 16.8% at 1.86 Å resolution. The molecular 2‐fold axis of the dimer is local but very close to a possible crystallographic 2‐fold axis; the slight asymmetry could be rationalized from the packing contacts. The 2 crystallographically independent subunits of the dimer are virtually identical, yielding no structural clue on possible cooperativity. The structure was compared with the well‐known structure of the homologous enzyme from human erythrocytes with 52% sequence identity. Significant differences were found at the dimer interface, where the human enzyme has a disulfide bridge, whereas the E. coli enzyme has an antiparallel β‐sheet connecting the subunits. The differences at the glutathione binding site and in particular a deformation caused by a Leu‐Ile exchange indicate why the E. coli enzyme accepts trypanothione much better than the human enzyme. The reported structure provides a frame for explaining numerous published engineering results in detail and for guiding further ones.
Biological meaning, statistical significance, and classification of local spatial similarities in nonhomologous proteins
Authors: Alexandrov Nickolai N. , Gō Nobuhiro
Published: July 2010

We have completed an exhaustive search for the common spatial arrangements of backbone fragments (SARFs) in nonhomologous proteins. This type of local structural similarity, incorporating short fragments of backbone atoms, arranged not necessarily in the same order along the polypeptide chain, appears to be important for protein function and stability. To estimate the statistical significance of the similarities, we have introduced a similarity score. We present several locally similar structures, with a large similarity score, which have not yet been reported. On the basis of the results of pairwise comparison, we have performed hierarchical cluster analysis of protein structures. Our analysis is not limited by comparison of single chains but also includes complex molecules consisting of several subunits. The SARFs with backbone fragments from different polypeptide chains provide a stable interaction between subunits in protein molecules. In many cases the active site of enzymes is located at the same position relative to the common SARFs, implying a function of the certain SARFs as a universal interface of the protein‐substrate interaction.
Native disulfide bonds greatly accelerate secondary structure formation in the folding of lysozyme
Authors: Goldberg Michel E. , Guillou Yvonne
Published: July 2010

To assess the respective roles of local and long‐range interactions during protein folding, the influence of the native disulfide bonds on the early formation of secondary structure was investigated using continuous‐flow circular dichroism. Within the first 4 ms of folding, lysozyme with intact disulfide bonds already had a far‐UV CD spectrum reflecting large amounts of secondary structure. Conversely, reduced lysozyme remained essentially unfolded at this early folding time. Thus, native disulfide bonds not only stabilize the final conformation of lysozyme but also provide, in early folding intermediates, the necessary stabilization that favors the formation of secondary structure.
Folding of the multidomain human immunodeficiency virus type‐I integrase
Authors: Grandgenett Duane P. , Goodarzi Goodarz
Published: July 2010

Protein folding conditions were established for human immunodeficiency virus integrase (IN) obtained from purified bacterial inclusion bodies. IN was denatured by 6 M guanidine · HCl–5 mM dithiothreitol, purified by gel filtration, and precipitated by ammonium sulfate. The reversible solvation of precipitated IN by 6 M guanidine · HCl allowed for wide variation of protein concentration in the folding reaction. A 6‐fold dilution of denatured IN by 1 M NaCl buffer followed by dialysis produced enzymatically active IN capable of 3′ OH end processing, strand transfer, and disintegration using various human immunodeficiency virus‐1 (HIV‐1) long terminal repeat DNA substrates. The specific activities of folded IN preparations for these enzymatic reactions were comparable to those of soluble IN purified directly from bacteria. The subunit composition and enzymatic activities of IN were affected by the folding conditions. Standard folding conditions were defined in which monomers and protein aggregates sedimenting as dimers and tetramers were produced. These protein aggregates were enzymatically active, whereas monomers had reduced strand transfer activity. Temperature modifications of the folding conditions permitted formation of mainly monomers. Upon assaying, these monomers were efficient for strand transfer and disintegration, but the oligomeric state of IN under the conditions of the assay is indeterminate. Our results suggest that monomers of the multidomain HIV‐1 IN are folded correctly for various catalytic activities, but the conditions for specific oligomerization in the absence of catalytic activity are undefined.
Monte Carlo study of the effect of β2‐microglobulin on the binding cleft of the HLA‐A2 complex
Authors: Bouzida Djamal , Garnier Jean , Brower Richard , Cornette James , Delisi Charles
Published: July 2010

Peptide recognition by class I products of the major histocompatibility complex requires association of the class I heavy chain with β2‐microglobulin. We present results of Monte Carlo simulations of the β‐pleated sheet floor of the human class I MHC molecule, HLA‐A2, with and without β2‐microglobulin. We find a significant effect of β2‐microglobulin on the side chains of residues near a region that would accommodate the C‐terminus of a bound peptide. By modeling simultaneously each loop and its neighboring strand at either end of the class I cleft, we find that β2‐microglobulin restricts the conformational space of residues that are central to binding peptides. The effect is most pronounced for R97 and H114 and somewhat less important for Y99 and Y116, the latter forming strong hydrogen bonds with neighboring residues in the heavy chain itself.
Harmonic and anharmonic aspects in the dynamics of BPTI: A normal mode analysis and principal component analysis
Authors: Hayward Steven , Kitao Akio , Gō Nobuhiro
Published: July 2010

A comparison is made between a 200‐ps molecular dynamics simulation in vacuum and a normal mode analysis on the protein bovine pancreatic trypsin inhibitor (BPTI) in order to elucidate the dual aspects of harmonicity and anharmonicity in the dynamics of proteins. The molecular dynamics trajectory is analyzed using principal component analysis, an effective harmonic analysis suited for comparison with the results from the normal mode analysis. The results suggest that the first principal component shows qualitatively different behavior from higher principal components and is associated with apparent barrier crossing events on an anharmonic conformational energy surface. The higher principal components appear to have probability distributions that are well approximated by Gaussians, indicating harmonicity. Eliminating the contribution from the first principal component reveals a great deal of correspondence between the 2 methods. This correspondence, however, involves a factor of 2, as the variances of the distributions of the higher principal components are, on average, roughly twice those found from the normal mode analysis. A model is proposed to reconcile these results with those from previous analyses.
Thermodynamics of staphylococcal nuclease denaturation. I. The acid‐denatured state
Authors: Carra John H. , Anderson Elizabeth A. , Privalov Peter L.
Published: July 2010

Using high‐sensitivity differential scanning calorimetry, we reexamined the thermodynamics of denaturation of staphylococcal nuclease. The denaturational changes in enthalpy and heat capacity were found to be functions of both temperature and pH. The denatured state of staphylococcal nuclease at pH 8.0 and high temperature has a heat capacity consistent with a fully unfolded protein completely exposed to solvent. At lower pH values, however, the heat capacity of the denatured state is lower, resulting in a lower ΔCp and ΔH for the denaturation reaction. The acid‐denatured protein can thus be distinguished from a completely unfolded protein by a defined difference in enthalpy and heat capacity. Comparison of circular dichroism spectra suggests that the low heat capacity of the acid‐denatured protein does not result from residual helical secondary structure. The enthalpy and heat capacity changes of denaturation of a less stable mutant nuclease support the observed dependence of ΔH on pH.
A 70‐amino acid zinc‐binding polypeptide fragment from the regulatory chain of aspartate transcarbamoylase causes marked changes in the kinetic mechanism of the catalytic trimer
Authors: Zhou Bin‐Bing , Waldrop Grover L. , Lum Lawrence , Schachman H. K.
Published: July 2010

Interaction between a 70‐amino acid and zinc‐binding polypeptide from the regulatory chain and the catalytic (C) trimer of aspartate transcarbamoylase (ATCase) leads to dramatic changes in enzyme activity and affinity for active site ligands. The hypothesis that the complex between a C trimer and 3 polypeptide fragments (zinc domain) is an analog of R state ATCase has been examined by steady‐state kinetics, heavy‐atom isotope effects, and isotope trapping experiments. Inhibition by the bisubstrate ligand, N‐(phosphonacetyl)‐L‐aspartate (PALA), or the substrate analog, succinate, at varying concentrations of substrates, aspartate, or carbamoyl phosphate indicated a compulsory ordered kinetic mechanism with carbamoyl phosphate binding prior to aspartate. In contrast, inhibition studies on C trimer were consistent with a preferred order mechanism. Similarly, 13C kinetic isotope effects in carbamoyl phosphate at infinite aspartate indicated a partially random kinetic mechanism for C trimer, whereas results for the complex of C trimer and zinc domain were consistent with a compulsory ordered mechanism of substrate binding. The dependence of isotope effect on aspartate concentration observed for the Zn domain‐C trimer complex was similar to that obtained earlier for intact ATCase. Isotope trapping experiments showed that the compulsory ordered mechanism for the complex was attributable to increased “stickiness” of carbamoyl phosphate to the Zn domain‐C trimer complex as compared to C trimer alone. The rate of dissociation of carbamoyl phosphate from the Zn domain‐C trimer complex was about 10–2 that from C trimer. Additional evidence for a change in binding of carbamoyl phosphate as a result of interaction of zinc domain and C trimer was obtained from the pH profile for Kia for carbamoyl phosphate. Whereas participation of only 1 protonated residue is implicated in binding of carbamoyl phosphate to C trimer, 2 residues must be ionized for binding to the Zn domain‐C trimer complex.
Identification of glutamate 344 as the catalytic residue in the active site of pig heart CoA transferase
Authors: Rochet Jean‐Christophe , Bridger William A.
Published: July 2010

The enzyme CoA transferase (succinyl‐CoA:3‐ketoacid coenzyme A transferase [3‐oxoacid CoA transferase], EC 2.8.3.5) is essential for the metabolism of ketone bodies in the mammalian mitochondrion. It is known that its catalytic mechanism involves the transient thioesterification of an active‐site glutamate residue by CoA. As a means of identifying this glutamate within the sequence, we have made use of a fortuitous autolytic fragmentation that occurs at the active site when the enzyme‐CoA covalent intermediate is heated. The presence of protease inhibitors has no effect on the extent of cleavage detectable by SDS‐PAGE, supporting the view that this fragmentation is indeed autolytic. This fragmentation can be carried out on intact CoA transferase, as well as on a proteolytically nicked but active form of the enzyme. Because the resulting C‐terminal fragment is blocked at its N‐terminus by a pyroglutamate moiety, it is not amenable to direct sequencing by the Edman degradation method. As an alternative, we have studied a peptide (peptide D) generated specifically by autolysis of the nicked enzyme and predicted to have an N‐terminus corresponding to the site of proteolysis and a C‐terminus determined by the site of autolysis. This peptide was purified by reversed‐phase HPLC and subsequently characterized by electrospray mass spectrometry. We have obtained a mass value for peptide D, from which it can be deduced that glutamate 344, known to be conserved in all sequenced CoA transferases, is the catalytically active amino acid. This information should prove useful to future mutagenesis work aimed at better understanding the active‐site structure and catalytic mechanism of CoA transferase.
Interleukin‐1βconverting enzyme: A novel cysteine protease required for IL‐1β production and implicated in programmed cell death
Authors: Thornberry Nancy A. , Molineaux Susan M.
Published: July 2010

Interleukin‐1β converting enzyme is the first member of a new class of cysteine proteases. The most distinguishing feature of this family is a nearly absolute specificity for cleavage at aspartic acid. This enzyme has been the subject of intense research because of its role in the production of IL‐1β, a key mediator of inflammation. These studies have culminated in the design of potent inhibitors and determination of its crystal structure. The structure secures the relationship of the enzyme to CED‐3, the product of a gene required for programmed cell death in Caenorhabditis elegans, suggesting that members of this family function in cell death in vertebrates.
Differentiation of peptide molecular recognition by phospholipase Cγ‐1 Src homology‐2 domain and a mutant Tyr phosphatase PTP1bC215S
Authors: Maclean Derek , Sefler Andrea M. , Zhu Guochang , Decker Stuart J. , Saltiel Alan R. , Singh Juswinder , Mcnamara Dennis , Dobrusin Ellen M. , Sawyer Tomi K.
Published: July 2010

Activated epidermal growth factor receptor (EGFR) undergoes autophosphorylation on several cytoplasmic tyrosine residues, which may then associate with the src homology‐2 (SH2) domains of effector proteins such as phospholipase Cγ‐1 (PLCγ‐1). Specific phosphotyrosine (pTyr)‐modified EGFR fragment peptides can inhibit this intermolecular binding between activated EGFR and a tandem amino‐ and carboxy‐terminal (N/C) SH2 protein construct derived from PLCγ‐1. In this study, we further explored the molecular recognition of phosphorylated EGFR988–998 (Asp‐Ala‐Asp‐Glu‐pTyr‐Leu‐Ile‐Pro‐Gln‐Gln‐Gly, I) by PLCγ‐1 N/C SH2 in terms of singular Ala substitutions for amino acid residues N‐ and C‐terminal to the pTyr (P site) of phosphopeptide I. Comparison of the extent to which these phosphopeptides inhibited binding of PLCγ‐1 N/C SH2 to activated EGFR showed the critical importance of amino acid side chains at positions P+2 (Ile994), P+3 (Pro995), and P+4 (Gln996). Relative to phosphopeptide 1, multiple Ala substitution throughout the N‐terminal sequence, N‐terminal truncation, or dephosphorylation of pTyr each resulted in significantly decreased binding to PLCγ‐1 N/C SH2. These structure‐activity results were analyzed by molecular modeling studies of the predicted binding of phosphopeptide 1 to each the N‐ and C‐terminal SH2 domains of PLCγ‐1. In the case of the PLCγ‐1 C‐terminal SH2, the coordinates of a phosphopeptide SH2 domain complex recently determined by NMR studies (Pascal SM, et al., 1994, Cell 77:461–472) were used directly, whereas the N‐terminal SH2 domain model was developed from homology model building using the NMR coordinates of the PLCγ‐1 C‐terminal SH2 as a template. Relative to PLCγ‐1 N/C SH2 domain molecular recognition, the binding affinities of phosphorylated EGFR988–998 and its Ala‐scan analog series to a catalytically inert mutant protein tyrosine phosphatase, PTPlbC215S, showed only a slight loss in binding at the P+1 (Leu) site. Therefore, peptide molecular recognition by two functionally distinct signal transduction proteins, PLCγ‐1 and PTP1b, may be differentiated in terms of systematic chemical modifications of EGFR988_998 and comparative structure‐activity analysis.
Thermodynamics of heme‐induced conformational changes in hemopexin: Role of domain‐domain interactions
Authors: Wu Ming‐Lei , Morgan William T.
Published: July 2010

Hemopexin is a serum glycoprotein that binds heme with high affinity and delivers heme to the liver cells via receptor‐mediated endocytosis. A hinge region connects the two non‐disulfide‐linked domains of hemopexin, a 35‐kDa N‐terminal domain (domain I) that binds heme, and a 25‐kDa C‐terminal domain (domain II). Although domain II does not bind heme, it assumes one structural state in apo‐hemopexin and another in heme‐hemopexin, and this change is important in facilitating the association of heme‐hemopexin with its receptor. In order to elucidate the structure and function of hemopexin, it is important to understand how structural information is transmitted to domain II when domain I binds heme. Here we report a study of the protein‐protein interactions between domain I and domain II using analytical ultracentrifugation and isothermal titration calorimetry. Sedimentation equilibrium analysis showed that domain I associates with domain II both in the presence and absence of heme with Kd values of 0.8 μM and 55 μM, respectively. The interaction between heme‐domain I and domain II has a calorimetric enthalpy of +11 kcal/mol, a heat capacity (ΔCP) of ‐720 cal/mol‐K, and a calculated entropy of +65 cal/mol‐K. By varying the temperature of the centrifugation equilibrium runs, a van't Hoff plot with an apparent change in enthalpy (ΔH) of ‐3.6 kcal/mol and change in entropy (AS) of +8.1 cal/mol‐K for the association of apo‐domain I with domain II was obtained. The association is, however, not strongly dependent on ionic strength. The values of the thermodynamic parameters obtained suggest that different types of interactions predominate in the presence and absence of heme: hydrophobic interactions between heme‐domain I and domain II and likely a balanced mix of different forces (e.g., van der Waals and hydrophobic interactions) between apodomain I and domain II.
Mutations of surface residues in Anabaena vegetative and heterocyst ferredoxin that affect thermodynamic stability as determined by guanidine hydrochloride denaturation
Authors: Hurley John K. , Caffrey Michael S. , Markley John L. , Cheng Hong , Xia Bin , Chae Young Kee , Holden Hazel M. , Tollin Gordon
Published: July 2010

The stability properties of oxidized wild‐type (wt) and site‐directed mutants in surface residues of vegetative (Vfd) and heterocyst (Hfd) ferredoxins from Anabaena 7120 have been characterized by guanidine hydrochloride (Gdn‐HCl) denaturation. For Vfd it was found that mutants E95K, E94Q, F65Y, F65W, and T48A are quite similar to wt in stability. E94K is somewhat less stable, whereas E94D, F65A, F65I, R42A, and R42H are substantially less stable than wt. R42H is a substitution found in all Hfds, and NMR comparison of the Anabaena 7120 Vfd and Hfd showed the latter to be much less stable on the basis of hydrogen exchange rates (Chae YK, Abildgaard F, Mooberry ES, Markley JL, 1994, Biochemistry 33:3287–3295); we also find this to be true with respect to Gdn‐HCl denaturation. Strikingly, the Hfd mutant H42R is more stable than the wt Hfd by precisely the amount of stability lost in Vfd upon mutating R42 to H (2.0 kcal/mol). On the basis of comparison of the X‐ray crystal structures of wt Anabaena Vfd and Hfd, the decreased stabilities of F65A and F65I can be ascribed to increased solvent exposure of interior hydrophobic groups. In the case of Vfd mutants E94K and E94D, the decreased stabilities may result from disruption of a hydrogen bond between the E94 and S47 side chains. The instability of the R42 mutants is also most probably due to decreased hydrogen bonding capabilities. Those F65 mutants showing diminished stability (i.e., F65A and F65I) have previously been shown (Hurley JK, et al., 1993b, Biochemistry 52:9346–9354) to be severely impaired kinetically in their electron transfer (ET) reaction with ferredoxin:NADP + reductase (FNR), a physiological reaction partner of Vfd. Mutants F65W and F65Y, which, as noted, are like wt in stability, also functioned like wt in the ET reaction with FNR (Hurley JK, et al., 1993a, J Am Chem Soc 115:11698–11701). Possible reasons for this correlation between ET properties of the F65 mutants and their conformational stabilities are discussed.
Simulations of CRP:(cAMP)2 in noncrystalline environments show a subunit transition from the open to the closed conformation
Authors: García Angel E. , Harman James G.
Published: July 2010

The CRP:cAMP complex functions as a transcription factor that facilitates RNA polymerase recognition of several bacterial promoters. Detailed crystal structure information is available for CRP:(cAMP)2 and for CRP:(cAMP)2 complexed with DNA. In the crystalline environment, CRP:(cAMP)2 subunits are asymmetrically related; one subunit has a closed conformation and the other has an open conformation. The CRP:(cAMP)2 complexed with DNA shows both subunits in a closed conformation. We have studied the molecular dynamics of CRP:(cAMP)2 in noncrystalline environments. CRP:(cAMP)2 was simulated for 625 ps in vacuo and for 140 ps in solution. The crystal structure of CRP:(cAMP)2 in the absence of DNA was used as the initial conformation. Molecule optimal dynamic coordinates (MODCs) (Garcia A, 1992, Phys Rev Lett 68:2696) were used to analyze protein conformations sampled during the course of the simulations. Two MODCs define a transition of the open subunit to a closed subunit conformation during the first 125 ps of simulation in vacuo; the resulting subunit conformation is similar to that observed in CRP:(cAMP)2:DNA crystals. Simulation of CRP:(cAMP)2 in solution showed that a transition from the open to the closed state also occurs when water is explicitly included in the calculations. These calculations suggest that the asymmetric conformation of CRP:(cAMP)2 is stabilized by crystal lattice interactions. The predicted solution conformation is more symmetric, with both subunits in a closed conformation.
Human protoporphyrinogen oxidase: Expression, purification, and characterization of the cloned enzyme
Authors: Dailey Tamara A. , Dailey Harry A.
Published: July 2010

Protoporphyrinogen oxidase (E.C.1.3.3.4) catalyzes the oxygen‐dependent oxidation of protoporphyrinogen IX to protoporphyrin IX. The enzyme from human placenta has been cloned, sequenced, expressed in Escherichia coli, purified to homogeneity, and characterized. Northern blot analysis of eight different human tissues show evidence for only a single transcript in all tissue types and the size of this transcript is approximately 1.8 kb. The human cDNA has been inserted into an expression vector for E. coli and the protein produced at high levels in these cells. The protein is found in both membrane and cytoplasmic fractions. The enzyme was purified to homogeneity in the presence of detergents using a metal chelate affinity column. The purified protein is a homodimer composed of subunits of a molecular weight of 51,000. The enzyme contains one noncovalently bound FAD per dimer, has a monomer extinction coefficient of 48,000 at 270 nm and contains no detectable redox active metals. The apparent Km and Kcat for protoporphyrinogen IX are 1.7 μM and 10.5 min−1, respectively. The enzyme does not use coproporphyrinogen III as a substrate and is inhibited by micromolar concentrations of the herbicide acifluorfen. Protein database searches reveal significant homology between protoporphyrinogen oxidase and monoamine oxidase.
Two mutations in recombinant Hb β F41(C7)Y, K82 (EF6)D show additive effects in decreasing oxygen affinityxs
Authors: Dumoulin A. , Kiger L. , Griffon N. , Vasseur C. , Kister J. , Génin P. , Marden M.C. , Pagnier J. , Poyart C.
Published: July 2010

Based on the properties of two low oxygen affinity mutated hemoglobins (Hb), we have engineered a double mutant Hb (rHb βYD) in which the βF41Y substitution is associated with K82D. Functional studies have shown that the Hb α2/β2(C7)F41Y exhibits a decreased oxygen affinity relative to Hb A, without a significantly increased auto‐oxidation rate. The oxygen affinity of the natural mutant βK82D (Hb Providence‐Asp) is decreased due to the replacement of two positive charges by two negative ones at the main DPG‐binding site. The functional properties of both single mutants are interesting in the view of obtaining an Hb‐based blood substitute, which requires: (1) cooperative oxygen binding with an overall affinity near 30 mm Hg at half saturation, at 37 °C, and in the absence of 2,3 diphosphoglycerate (DPG), and (2) a slow rate of autooxidation in order to limit metHb formation. It was expected that the two mutations were at a sufficient distance (20 Å) that their respective effects could combine to form low oxygen affinity tetramers. The double mutant does display additive effects resulting in a fourfold decrease in oxygen affinity; it can insure, in the absence of DPG, an oxygen delivery to the tissues similar to that of a red cell suspension in vivo at 37 °C. Nevertheless, the rate of autooxidation, 3.5‐fold larger than that of Hb A, remains a problem.
Pressure‐induced perturbation of ANS‐apomyoglobin complex: Frequency domain fluorescence studies on native and acidic compact states
Authors: Bismuto Ettore , Irace Gaetano , Sirangelo Ivana , Gratton Enrico
Published: July 2010

The pressure dependence of the flexibility of the 8‐anilino‐1‐naphthalene sulfonate (ANS)‐apomyoglobin complex was investigated in the range between atmospheric pressure and 2.4 kbar by frequency domain fluorometry. We examined two structural states: native and acidic compact. The conformational dynamics of the ANS‐apomyoglobin complex were deduced by studying the emission decay of ANS, which can form a noncovalent complex with the apoprotein in both the native and the acidic compact forms. Because the free fluorophore has a very short lifetime (less than 75 ps), its contribution can be separated from the long‐lived emission. The latter arises from ANS molecules bound to the protein and provides information on the structural and dynamic characteristics of the macromolecule. The fluorescence emission decay of the ANS‐apomyoglobin complex at neutral pH has a broad fluorescence lifetime distribution (width at half‐maximum = 4.1 ns). The small changes in the fluorescence distribution parameters that occur with changes in pressure indicate that the ANS‐apomyoglobin complex at neutral pH holds its compactness even at 2.4 kbar. A small contraction of molecular volume has been detected at low pressure, followed by a slight swelling with an increase in flexibility at higher pressures. The heterogeneity of ANS fluorescence in the acidic compact state of apomyoglobin is even greater than that in the native form (distribution width = 10 ns); moreover, the acidic compact state appears more expanded and accessible to solvent molecules than the native state, as suggested by the distribution center, which is 11 ns for the former and 19 ns for the latter. The lifetime distribution center remains constant with increasing pressure, which suggests that no other binding site is formed at high pressure.
Dynamics of the three methionyl side chains of Streptomyces subtilisin inhibitor. Deuterium NMR studies in solution and in the solid state
Authors: Tamura Atsuo , Matsushita Michio , Naito Akira , Kojima Shuichi , Miura Kin‐Ichiro , Akasaka Kazuyuki
Published: July 2010

Streptomyces subtilisin inhibitor (SSI) contains three methionine residues in a subunit: two (at positions 73 and 70) in the crucial enzyme‐recognition sites P1 and P4, respectively, and one (Met 103) in the hydrophobic core. The motions of the side chains of these three Met residues and the changes in mobility on binding with subtilisin were studied by deuterium NMR spectroscopy in solution and in crystalline and powder solids. For this purpose, the wild‐type SSI was deuterium‐labeled at the methyl groups of all three Met residues, and three artificial mutant proteins were labeled at only one specific Met methyl group each. In solution, for methionines 73 and 70, the effective correlation times were only 0.8–1.0 × 10−10 sindicating that the two side chains on the surface fluctuate almost freely. On formation of a complex with subtilisin, however, these high mobilities were quenched, giving a correlation time of 1.1 ± 10−8 s for the side chains of methionines 70 and 73. The correlation time of Met 103, located in the hydrophobic core, was at least 1.0 ± 10−8 s in free SSI, showing that its side chain motion is highly restricted. The nature of the internal motions of the three Met side chains was examined in more detail by deuterium NMR spectroscopy of powder and crystalline samples. The spectral patterns of the powder samples depended critically on hydration: immediately after lyophilization, the side‐chain motions of the three Met residues were nearly quenched. With gradual hydration to 0.20 gram of water per gram protein‐water, the orientational fluctuation of the methyl axes of methionines 70 and 73 was selectively enhanced in both amplitude and frequency (to about 1 MHz) and, at nearly saturating hydration (0.60 gram of water per gram protein‐water), became extremely high in amplitude and frequency (>10 MHz). In contrast, the polycrystalline wild‐type SSI spectrum showed fine structures, reflecting characteristic motions of the Met side chains. The polycrystalline spectrum could be reproduced reasonably well by the same motion models and parameters used to simulate the powder spectrum at the final level of hydration, suggesting that the side‐chain motions are similar in the fully hydrated powder and in crystals. Spin‐lattice relaxation measurements gave evidence that, even in crystals, the methyl axes of all three Met residues undergo rapid motions with correlation times between 10−8 and 10−10 s, comparable to the correlation times in solution. Finally, in the hydrated stoichiometric complex of SSI with subtilisin BPN' in the solid state, large‐amplitude motions are absent, but the side chains of methionines 70 and/or 73 are likely to have small‐amplitude motions.
Identification of the residues responsible for the alkaline inhibition of Cu, Zn superoxide dismutase: A site‐directed mutagenesis approach
Authors: Polticelli Fabio , Desideri Alessandro , Battistoni Andrea , Rotilio Giuseppe , O'Neill Peter
Published: July 2010

The catalytic rate of wild type, two single (Lys 120 → Leu, Lys 134 → Thr), and one double (Lys 120 → Leu‐Lys 134 → Thr) mutants of Xenopus laevis B Cu, Zn superoxide dismutase has been studied by pulse radiolysis as a function of pH. The pH dependence curve of the activity of the wild‐type enzyme can be deconvoluted by two deprotonation equilibria, at pH 9.3 (pK1) and at pH 11.3 (pK2). Catalytic rate measurements on single and double mutants indicate that pK1 is mainly due to the deprotonation of Lys 120 and Lys 134, with only a minor contribution from other surface basic residues, whereas pK2 is due to titration of the invariant Arg 141, likely coupled to deprotonation of the copper‐bound water molecule. Accordingly, Brownian dynamics simulations carried out as a function of pH reproduce well the pH dependence of the catalytic rate, when the experimentally determined pKs are assigned to Lys 120, Lys 134, and Arg 141.
Equilibrium dissociation and unfolding of the dimeric human papillomavirus strain‐16 E2 DNA‐binding domain
Authors: Mok Yu‐Keung , Bycroft Mark , Gay Gonzalo De Prat , Jonathan Butler P.
Published: July 2010

The equilibrium unfolding reaction of the C‐terminal 80‐amino‐acid dimeric DNA‐binding domain of human papillomavirus (HPV) strain 16 E2 protein has been investigated using fluorescence, far‐UV CD, and equilibrium sedimentation. The stability of the HPV‐16 E2 DNA‐binding domain is concentration‐dependent, and the unfolding reaction is well described as a two‐state transition from folded dimer to unfolded monomer. The conformational stability of the protein, ΔGH2O, was found to be 9.8 kcal/mol at pH 5.6, with the corresponding equilibrium unfolding/dissociation constant, Ku, being 6.5 → 10−8 M. Equilibrium sedimentation experiments give a Kd of 3.0 → 10−8 M, showing an excellent agreement between the two different techniques. Denaturation by temperature followed by the change in ellipticity also shows a concomitant disappearance of secondary and tertiary structures. The Ku changes dramatically at physiologically relevant pH's: with a change in pH from 6.1 to 7.0, it goes from 5.5 → 10−8 M to 4.4 → 1010 M. Our results suggest that, at the very low concentration of protein where DNA binding is normally measured (e.g., 10−11 M), the protein is predominantly monomeric and unfolded. They also stress the importance of the coupling between folding and DNA binding.
Posttranslational processing of recombinant human interferon‐γ in animal expression systems
Authors: Freedman Robert B. , Jenkins Nigel , James David C. , Goldman Merlin H. , Hoare Michael , Oliver Ronald W. A. , Green Brian N.
Published: July 2010

We have characterized the heterogeneity of recombinant human interferon‐γ (IFN‐γ) produced by three expression systems: Chinese hamster ovary cells, the mammary gland of transgenic mice, and baculovirus‐infected Spodoptera frugiperda (Sf9) insect cells. Analyses of whole IFN‐γ proteins by electrospray ionization‐mass spectrometry (ESI‐MS) from each recombinant source revealed heterogeneous populations of IFN‐γ molecules resulting from variations in N‐glycosylation and C‐terminal polypeptide cleavages. A series of more specific analyses assisted interpretation of maximum entropy deconvoluted ESI‐mass spectra of whole IFN‐γ proteins; MALDI‐MS analyses of released, desialylated N‐glycans and of deglycosylated IFN‐γ polypeptides were combined with analyses of 2‐aminobenzamide labeled sialylated N‐glycans by cation‐exchange high‐performance liquid chromatography. These analyses enabled identification of specific polypeptide cleavage sites and characterization of associated N‐glycans. Production of recombinant IFN‐γ in the mammalian expression systems yielded polypeptides C‐terminally truncated at dibasic amino acid sites. Mammalian cell derived IFN‐γ molecules displayed oligosaccharides with monosaccharide compositions equivalent to complex, sialylated, or high‐mannose type N‐glycans. In contrast, IFN‐γ derived from baculovirus‐infected Sf9 insect cells was truncated further toward the C‐terminus and was associated with neutral (nonsialylated) N‐glycans. These data demonstrate the profound influence of host cell type on posttranslational processing of recombinant proteins produced in eukaryotic systems.
BSTI, a trypsin inhibitor from skin secretions of bombina bombina related to protease inhibitors of nematodes
Authors: Mignogna Giuseppina , Pascarella Stefano , Amiconi Gino , Barra Donatella , Wechselberger Christian , Hinterleitner Christa , Mollay Christa , Kreil Günther
Published: July 2010

From skin secretions of the European frog Bombina bombina, a new peptide has been isolated that contains 60 amino acids, including 10 cysteine residues. Its sequence was determined by automated Edman degradation and confirmed by analysis of the cDNA encoding the precursor. A search in the databanks demonstrated that the pattern of cysteine residues in this skin peptide is similar to the ones found in protease inhibitors from Ascaris and in a segment of human von Willebrand factor. The 3D structure of the trypsin inhibitor from Ascaris suum could be used as a template to build a model of the amphibian peptide. In addition, we have demonstrated that this constituent of skin secretion is indeed an inhibitor of trypsin and thrombin, with Ki values in the range of 0.1 to 1 μM. The new peptide was thus named BSTI for Bombina skin trypsin/thrombin inhibitor.