bluuebunny/crossref_metadata_2025 huggingface dataset:
A very short peptide makes a voltage‐dependent ion channel: The critical length of the channel domain of colicin E1
Authors: Liu Q. R. , Crozel V. , Levinthal F. , Slatin S. , Finkelstein A. , Levinthal C.
Published: May 2005

Cleavage of colicin E1 molecules with a variety of proteases or with cyanogen bromide (CNBr) generates COOH‐terminal fragments which have channel‐forming activity similar to that of intact colicin in planar lipid bilayer membranes. The smallest channel‐forming fragment obtained by CNBr cleavage of the wild‐type molecule consists of the C‐terminal 152 amino acids. By the use of oligonucleotide‐directed mutagenesis, we have made nine mutants along this 152 amino acid peptide, in which an amino acid was replaced by methionine in order to create a new CNBr cleavage site. The smallest of the CNBr‐cleaved C‐terminal fragments with channel‐forming activity, in planar bilayer membranes, was generated by cleavage at new Met position 428 and has 94 amino acids, whereas a 75 amino acid peptide produced by cleavage of a new Met at position 447 did not have channel activity. The NH2‐terminus of the channel‐forming domain of colicin E1 appears therfore to lie between residues 428 and 447. Since, however, the last six C‐terminal residues of the colicin can be removed without changing activity, the number of amino acids necessary to form the channel is 88 or less. In addition, the unique Cys residue in colicin E1 was replaced by Gly, and nine mutants were then made with Cys placed at sequential locations along the peptide for eventual use as sulfhydryl attachment sites to determine the local environment of the replaced amino acid. In the course of making 21 mutants, eight charged residues have been replaced by uncharged Met or Cys without changing the biological activity of the intact molecule.

It has been proposed previously that the conformation of the colicin E1 channel is a barrel formed from five or six α‐helices, each having 20 amino acids spanning the membrane and two to four residues making the turn at the boundary of the membrane. Our finding that 88 amino acids can make an active channel, combined with recently reported stoichiometric evidence that the channel is a monomer excludes this model and adds significant constraints which can be used in building a molecular model of the channel.
The DNA replication inhibitor microcin B17 is a forty‐three‐amino‐acid protein containing sixty percent glycine
Authors: Davagnino Juan , Herrero Marta , Furlong Dierdre , Moreno Felipe , Kolter Roberto
Published: May 2005

Microcin B17 is a low‐molecular‐weight protein that inhibits DNA replication in a number of enteric bacteria. It is produced by bacterial strains which harbor a 70‐kilobase plasmid called pMccB17. Four plasmid genes (named mcbABCD) are required for its production. The product of the mcbA gene was identified by labelling minicells. The mcbA gene product was slightly larger when a mutation in any of the other three production genes was present. This indicates that these genes are involved in processing the primary mcbA product to yield the active molecule. The mcbA gene product predicted from the nucleotide sequence has 69 amino acids including 28 glycine residues. Microcin B17 was extracted from the cells by boiling in 100 mM acetic acid, 1 mM EDTA, and purified to homogeneity in a single step by high‐performance liquid chromatography through a C18 column. The N‐terminal amino acid sequence and amino acid composition demonstrated that mcbA is the structural gene for microcin B17. The active molecule is a processed product lacking the first 26 N‐terminal residues. The 43 remaining residues include 26 glycines. While microcin B17 is an exported protein, the cleaved N‐terminal peptide does not have the characteristic properties of a “signal sequence,” which suggests that it is secreted by a mechanism different from that used by most secreted proteins of E. coli.
Primary structure of the reaction center from Rhodopseudomonas sphaeroides
Authors: Williams J. C. , Steiner L. A. , Feher G.
Published: May 2005

The reaction center is a pigmentprotein complex that mediates the initial photochemical steps of photosynthesis. The amino‐terminai sequences of the L, M, and H subunits and the nucleotide and derived amino acid sequences of the L and M structural genes from Rhodopseudomonas sphaeroides have previously been determined. We report here the sequence of the H subunit, completing the primary structure determination of the reaction center from R. sphaeroides. The nucleotide sequence of the gene encoding the H subunit was determined by the dideoxy method after subcloning fragments into single‐stranded M13 phage vectors. This information was used to derive the amino acid sequence of the corresponding polypeptide. The termini of the primary structure of the H subunit were established by means of the amino and carboxy terminal sequences of the polypeptide. The data showed that hte H subunit is composed of 260 residues, corresponding to a molecular weight of 28,003. A molecular weight of 100,858 for the reaction center was calculated from the primary structures of the subunits and the cofactors. Examination of the genes encoding the reaction center shows that the codon usage is strongly bviased towards codons ending in G and C. Hydropathy analysis of the H subunit sequence reveals one stretch opf hydrophobic residues near the amino terminus; the L and M subunits contain five such stretches. From a comparison of the sequences of homologous proteins found in bacterial reaction centers and photosystem II of plants, an evolutionary tree was contructed. The analysis of evolutionary relationships showed that the L and M subunits of reaction centers and D1 and D2 proteins of photosystem II are descended from a common ancestor, and that the rate of change in these proteins was much higher in the first billion years after the divergence of the reaction center and photosystem II than in the subsequent billion years represented by the divergence of the species containing these proteins.
Hybrid immunoglobulin isotypes of identical specificity produced by genetic recombination in Escherichia coli and expression in lymphoid cells
Authors: Schneider William P. , Oi Vernon T. , Yanofsky Charles
Published: May 2005

We have produced a series of hybrid IgG1‐IgG2a mouse immunoglobulins with identical light chains (L) and variable regions to facilitate the identification of structural features associated with functional differnces between immunoglobulin isotypes. Hybrid heavy chain (H) constant region gene segments were generated by genetic recombination in Escherichia coli between plasmids carrying mouse γ1 and γ2a gene segments. Crossovers occured through out these segments although the frequency was highest in regions of high nucleotide sequence homology. Eleven variant immunoglobulins produced by transfected hybridoma cell lines are assembled into H2L2 tetramers and properly glycosylated. In addition, all 11 immunoglbulins have identical antigen combining sites specific for the fluorescent hapten ε‐dansyll‐L‐lysine. Protein A binding was used as probe of the structural integrity of the Fc portion of the variant antibodies. Differeneces in protein A binding between IgG1 and IgG2a appear to be due to amino acid differances at postions 252 (Thr→Met) and 254 (Thr→Ser) of the heavy chain (EU numbering).
Prediction of the tertiary structure of the α‐subunit of tryptophan synthase
Authors: Hurle Mark R. , Matthews C. Robert , Cohen Fred E. , Kuntz I.D. , Toumadje Arazdordi , Johnson W. Curtis
Published: May 2005

The tertiary structure of the α‐subunit of tryptophan synthase was proposed using a combination of experimental data and computational methods. The vacuum‐ultraviolet circular dichroism spectrum was used to assign the protein to the α/β‐class of supersecondary structures. The two‐domain structure of the α‐subunit (Miles et al.: Biochemistry 21:2586, 1982; Beasty and Matthews: Biochemistry 24:3547, 1985) eliminated consideration of a barrel structure and focused attention on a β‐sheet structure. An algorithm (Cohen et al.: Biochemistry 22:4894, 1983) was used to generate a secondary structure prediction that was consistent with the sequence data of the α‐subunit from five species. Three potential secondary structures were then packed into tertiary structures using other algorithms. The assumption of nearest neighbors from second‐site revertant data eliminated 97% of the possible tertiary structures; consideration of conserved hydrophobic packing regions on the β‐sheet eliminated all but one structure. The native structure is predicted to have a parallel β‐sheet flanked on both sides by α‐helices, and is consistent with the available data on chemical cross‐linking, chemical modification, and limited proteolysis. In addition, an active site region containing appropriate residues could be identified as well as an interface for β2‐subunit association. The ability of experimental data to facilitate the prediction of protein structure is discussed.
Lysine/fibrin binding sites of kringles modeled after the structure of kringle 1 of prothrombin
Authors: Tulinsky Alexander , Park Chang H. , Mao Boryeu , Llináas Miguel
Published: May 2005

The Lys binding site of kringle 1 and 4 (K1 and K4) of plasminogen (PG) has been modeled on the basis of the three‐dimensional structure of kringle 1 of prothrombin and 300‐ and 600‐MHz proton nuclear magnetic resonance observations. These structures were then compared to the corresponding regions of modeled kringle 1 and 2 of tissue plasminogen activator (PA). The coordinates of the modeled structures have been refined by energy minimization in the presence and absence of ϵ‐aminocaproic acid ligand in order basically to remove unacceptable van der Waals contacts. The binding site is characterized by an apparent dipolar surface, the polar parts of which are separated by a hydrophobic region of highly conserved aromatic residues. Zwitterionic ligands such as Lys and ϵ‐aminocaproic acid form ion pair interactions with Asp55 and Asp57 located on the dipolar surface; the latter are also conserved in all the Lys binding kringles. The cationic center of the dipolar surface is Arg71, in the case of PGK4, and is composed of Arg34 and Arg71 in PGK1. The doubly charged anionic/cationic interaction centers of the latter might account for the larger binding constants of PGK1 for like‐ligands but the modeling suggests that PGK4 might be kinetically faster in binding bulkier ligands. The binding site region of PAK2, which also binds Lys, resembles those of PGK1 and PGK4. Since PAK2 lacks both cationic center Arg residues, ligand carboxylate binding appears to be accomplished though an imidazolium ion of His64, which is located just below the outer surface of the kringle.
Model of a complex between the tetrahemic cytochrome c3 and the ferredoxin I from Desulfovibrio desulfuricans (Norway strain)
Authors: Cambillau C. , Frey M. , Mossé J. , Guerlesquin F. , Bruschi M.
Published: May 2005

A three‐dimensional model of an electron‐transfer complex between the tetrahemic cytochrome c3 and the ferredoxin I from the sulfatereducing bacterium Desulfovibrio desulfuricans (Norway strain) has been generated through computer graphics methods. The model is based on the known X‐ray structure of the cytochrome and on a model of the ferredoxin that has been derived through computer graphics modeling and energy minimization methods, from the X‐ray structure of the homologous ferredoxin from Peptococcus aerogenes. Four possible models of interaction between the two molecules were examined by bringing in close proximity each of the four hemes and the redox center (4Fe‐4S) of the ferredoxin and by optimizing the ion pairs interactions. One of these models shows by far the “best” structure in terms of charges, interactions, and complementary f the topology of the contact surfaces. In this complex, the distance between the iron atoms of the ferredoxin redox center and the hemic iron atom is 11.8 Å, which compares well with those found between redox centers in other complexes. The contact surface area between the two molecules is 170 Å2.
Crystallization and preliminary investigation of single crystals of deoxyuidine triphosphate nucleotidohydrolase from Escherichia coli
Authors: Cedergren‐Zeppezauer Eilia S. , Larsson Gunilla , Hoffmann Ingrid , Törnoos Karl W. , Al‐Karadaghi Salam , Nyman Per Olof
Published: May 2005

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase), an enzyme in the nucleotide metabolism that is a pyrophosphatase hydrolyzing dUTP, has been crystallized. The crystals belong to the trigonal space group R3 and diffract beyond 2 Å. The native dUTPase crystals and a mercury derivative are stable in the X‐ray beam and are suitable for a high resolution X‐ray structure analysis.
Homology of lysosomal enzymes and related proteins: Prediction of posttranslational modification sites including phosphorylation of mannose and potential epitopic and substrate binding sites in the α‐ and β‐subunits of hexosaminidases, α‐glucosidase, and rabbit and human isomaltase
Authors: Barnes Alison K. , Wynn Colin H.
Published: May 2005

Recently developed computer programs, including secondary structure and epitopic site predictions, have been used to align lysosomal proteins for maximum homology, based on conservative interchanges, and the aligned sequences have been searched for potential sites for posttranslational modification, glycosyaltion, and binding and catalysis of substrate. The homology and prediction of the posttranslational modification of the α‐ and β‐subunits of hexosaminidase is in good agreement with previous observations, and an explanation of the differing substrate specificities of the two subunits is advanced. We shows that the striking homology between α‐glucosidase and isomaltase is reflected in that apparent conversation of the active site in both enzymes. Nonhomologous regions have been examined in detail in a search for binding sited for glycogen and maltose, and two such sites have been tentatively identified. A highly redundant consensus sequence for the Phosphorylation of mannose in lysosomal proteins, YXX(Y, W, or F), is suggested.
Second virial coefficient of α‐crystallin
Authors: Wang Xiaowei , Bettelheim Frederick A.
Published: May 2005

Light scattering studies were performed on bovine α‐crystallin measuring the scattering intensities as a function of scattering angle, concentration, and temperature. The data yielded the molecular weight, radius of gyration, and second virial coefficient of α‐crystallin at different temperatures. The second virial coefficient increased with increasing temperature. Both the enthalpy and entropy of solution of α‐crystallin are positive. The Flory thetatemperature was found to be 271 K.
A protein structural motif that bends DNA
Authors: White Stephen W. , Appelt Krzysztof , Wilson Keith S. , Tanaka Isao
Published: May 2005

The prokaryotic protein HU, integration host factor (IHF) from Escherichia coli and transcription factor 1 (TF1) from bacteriophage SPO1 are closely related molecules. Biochemical results suggest that the role of these proteins is to bind and bend DNA. From the high‐resolution structure of HU, we propose a model for thisinteraction with DNA. Crucial amino acid differences between the proteins can be rationalized in terms of their different specific functions.
Characterization and autoprocessing of precursor and mature forms of human immunodeficiency virus type 1 (HIV 1) protease purified from Escherichia coli
Authors: Strickler James E. , Gorniak Joselina , Dayton Brian , Meek Thomas , Moore Michael , Magaard Victoria , Malinowski John , Debouck Christine
Published: May 2005

A recombinant plasmid encompassing the human immunodeficiency virus type 1 (HIV 1) protease coding sequence and flanking regions (Ala‐13 to Gly‐185 of the pol open reading frame) has been expressed in two distinct strains of Escherichia coli, AR58 and AR68. In the first strain, AR58, the primary translation product, a 25 kilodalton (kDa) precursor protein, is short‐lived and rapidly processes itself to the 11 kDa mature protease in vivo. In the second strain, AR68, the 25 kDa species isonly partially processed, and it, a 13 kDA intermediate, and the mature 11 kDA enzyme accumulate at a ratio of 3:4.5:2.5, respectively. The 11 kDa mature protease from AR58 and the 25 kDa precursor from AR68 have been purified to homogeneity. The yield of 11 kDa enzyme from AR58 is approximately 0.02 mg/g wet weight of E. coli cell pellet. The protease has both the expected NH2‐ and COOH‐terminal sequences. The yield of 25 kDa enzyme from AR68 is approximately 0.1 mg/g wet weight of E. coli cell pellet. In vitro, the 25 kDa precursor enzyme rapidly (t1/2≅ 9 min) processes itself into a species with a mass of ∼13kDa and a species with a mass of ∼11 kDa. Both of these latter species can be separated by RP‐HPLC, have the NH2‐terminal sequence expected for the mature protease, and are active. The 11 kDa enzyme from AR58 comigrates with the 11 kDa enzyme from AR68 on RP‐HPLC and SDS poly acrylamide gel electrophoresis. On extended incubation at 4°C at either neutral or acidic pH all species of the proteinexhibit further autodegradation at defined sequences. The availability of the mature, 11 kDa enzyme and the 25 kDa precursor will allow biochemical and physical studies on this critical viral enzyme.
Site‐directed mutagenesis of colicin E1 provides specific attachment sites for spin labels whose spectra are sensitive to local conformation
Authors: Todd A. Paul , Cong Jianping , Levinthal Francoise , Levinthal Cyrus , Hubell Wayne L.
Published: May 2005

Colicin E1 is an E. coli plasmid‐lencoded water‐soluble protein that spontaneously inserts into lipid membranes to form a voltage‐gated ion channel. We have employed a novel approach is which site‐directed mutagenesis is used to provide highly specific attachment points for nitroxide spin labels. A series of colicin mutants, differing only by the position of a single cysteine residue, were prepared and selectively labeled at that cysteine. A hydrophilic sequence (398–406) within the C‐terminal domain of the water‐soluble form of the protein was investigated and exhibited an electron paramagnetic resonancc (EPR) spectral periodicity strongly suggesting an amphiphilic α‐helix. After removal of the Nterminus of the protein with trypsin, the spectra for this sequence indicate increased label mobility and a more flexible structure.
Three‐dimensional structure of the neurotoxin ATX Ia from Anemonia sulcata in aqueous solution determined by nuclear magnetic resonance spectroscopy
Authors: Widmer Hans , Billeter Martin , Wüthrich Kurt
Published: May 2005

With the aid of 1H nuclear magnetic resonance (NMR) spectroscopy, the three‐dimensional structure in aqueous solution was determined for ATX Ia, which is a 46 residue polypeptide neurotoxin of the sea anemone Anemonia sulcata. The input for the structure calculations consisted of 263 distance constraints from nuclear Overhauser effects (NOE) and 76 vicinal coupling constants. For the structure calculation several new or ammended programs were used in a revised strategy consisting of five successive computational steps. First, the program HABAS was used for a complete search of all backbone and χ1 conformations that are compatible with the intraresidual and sequential NMR constraints. Second, using the program DISMAN, we extended this approach to pentapeptides by extensive sapling of al conformations that are consistent with the local and medium‐range NMR constraints. Both steps resulted in the definition of additional dihedral angle constraints and in stereospecific assignments for a number of β‐methylene groups. In the next two steps DISMAN was used to obtain a group of eight conformers that contain no significant residual violations of the NMR constraints or van der Waals contacts. Finally, these structures were subjected to restrained energy refinement with a modified version of the molecular mechanics module of AMBER, which in addition to the energy force field includes potentials for the NOCE distance constraints and the dihedral angle constraints. The average of the pairwise minimal RMS distances between the resulting refined conformers calculated for the well defined molecular core, which contains the backbone atoms of 35 residues and 20 interior side chains, is 1.5 ± 0.3 Å. This core is formed by a four‐stranded β‐sheet connected by two well‐defined loops, and there is an additional flexible loop consisting of the eleven residues 8‐18. The core of the protein is stabilized by three disulfide bridges, which are surrounded by hydrophobic residues and shielded on one side by hydrophilic residues.
Protein–drug interactions: Characterization of inhibitor binding in complexes of DHFR with trimethoprim and related derivatives
Authors: Fleischmann Stephen H. , Brooks Charles L.
Published: May 2005

Structural and thermodynamic interactions for the binding of trimethoprim and related congeners to the binary complex of diphydrofolate reductase (from chicken) and NADPH are explored using free energy simulation methods. Good agreement between structures from experimental X‐ray refinement and molecular dynamics simulations is found for the complexes. Agreement with thermodyanmic measurements is found as well. Our thermodynamic calculations suggest that entropic contributions and desolvation thermodynamics can play a crucial role in overall bindings, and that extreme care must be taken in the use of simple model building to rationalize or predict protein–drug binding.
Structure determination and refinment of Bacillus stearothermophilus lactate dehydrogenase
Authors: Piontek Klaus , Chakrabarti Pinakpani , Schär Hans‐Peter , Rossmann Michael G. , Zuber Herbert
Published: May 2005

Structures have been determined of Bacillus stearothermophilus “apo” and holo lactate dehydrogenase. The holo‐enzyme had been co‐crystallized with the activator fructose 1,6‐biosphosphate. The “apo” lactate dehydrogenase structure was solved by use of the known apo‐M4 dogfish lactate dehydrogenase molecule as a starting model. Phases were refined and extended from 4 Å resolution by means of the noncrystallographic molecular 222 symmetry. The R‐factor was reduced to 28.7%, using 2.8 Å resolution data, in a restrained least‐squares refnement in which the molecula rsymmetry was imposed as a constraint. A low occupancy of coenzyme was found in each of the four subunits of the “apo” enzyme.

Further refinement proceeded with the isomorphous holo‐enzyume from Bacillus Stearothermophilus. After removing the noncrystallographic constraints, the R‐factor dropped from 30.3% to a final value of 26.0% with a 0.019 Å and 1.7° r.m.s. deviation from idealized bond length and angles, respectively.

Two sulfate ions per subunit were included in the final model of the “apo” ‐from‐one at the substrate binding site and one close to the molecular P ‐axis near the location of the fructose 1,6‐bisphosphate activator. The final model of the holo‐enzyme incorporated two sulfate ions per subunit, one at the substrate binding site and another close to the R‐axis. One nicotinamide adenine dinucleotide coenzyme molecule per subunit and two fructose 1,6‐bisphosphate molecules per tetramer were also included. The phosphate positions of fructose 1,6‐bisphosphate are close to the sulfate ion near the P‐axis in the “apo” model.

This structure represents the first reported refined model of an allosteric activated lactate dehydrogenase. The structure of the activated holo‐enzyme showed far greater similarity to the ternary complex of dogfish M4 lactate dehydrogenase with incotinamide adenine dinucleotide and oxamate than to apo‐M4 dogfish lactate dehydrogenase. The conformations of nicotinamide adenine dinucleotide and fructose, 1,6‐bisphosphate were also analyzed.
Computer analysis of mutations that affect antibody specificity
Authors: Novotny Jiri , Bruccoleri Robert E. , Haber Eldgar
Published: May 2005

The mouse hybridoma cell line 40–150 scretes antibodies with high affinity towards the cardiac glycosides digoxin and digitoxin. A spontaneous mutant, 40–150 A2.4, produces and antibody which carries a single residue mutation, Ser → Arg, in its heavy chain (H94) and has an altered specificity. A second order mutant 40–150 A2.4 P.10, produces two antibody molecules, one the same as 40–150 A2.4, the other lacking two residues at the N‐terminus of its H chain, and having a specificity profile approaching that of 40–150 antibody. 1 The N‐terminus and the position H94 are distant from the antigen‐binding site of the antibody; thus, the structural basic of the specificity changes was not immediately clear. Approximate structures of the 40–150 antibody and its mutants were constructed in the computer, based on atomic coordinates of the homologous mouse antibody McPC 603. Using the program OCNGEN, the torsional space of the polypeptide backbone and side chains around position H94 was uniformly sampled, and the lowest energy conformations were analyzed in detail. The results indicate that when Arg‐H94 is substituted for Ser. Agr‐H94 can hydrogen bond to side chains of Asp‐H101, Arg‐L46, and Asp‐L55. The results in a change in the surface of the combining site which may account for the affinity changes. Deletion of the two N‐terminal residues increases solvent accessibility of Arg‐H94. The solvation may cause a hydrogen bond between Arg‐H94 and Asp‐H101 to be lost, restoring the structure to one similar to that of 40–150.
Model for the interaction of amphiphilic helices with troponin C and calmodulin
Authors: Strynadka Natalie C. J. , James Michael N. G.
Published: May 2005

Crystals of troponin are stabilized by an intermolecular interaction that involves the packing of helix A from the N‐terminal domain of one molecule onto the exposed hydrophobic cleft of the C‐terminal domain of a symmetry related molecules. Analysis of this molecular recognition interaction in troponin C suggests a possible mode for the binding of amphiphilic helical molecules to troponin C and to calmodulin. From the template provided by this troponin C packing, it has been possible to build a model of the contact region of mastoporan as it might be bound to the two Ca2+ binding proteins. A possible binding mode of melittin to calmodulin is also proposed. Although some of the characteristics of binding are similar for the two amphiphilic peptides, the increased length of melittin requires a significant bend in the calmodulin central helix similar to that suggested recently for the myosin light chain kinase calmodulin binding peptide (Persechini and Kretsinger: Journal of Cardiovascular Pharmacology 12:501–512, 1988). Not only are the hydrophobic interactions important in this model, but there are several favorable electrostatic interactions that are predicted as a result of the molecular modeling. The regions of troponin‐C and calmodulin to which amphiphilic helices bind are similar to the regions to which the neuroleptic drugs such as trifluoperazine have been predicted to bind (Strynadka and James: Proteins 3:1–17, 1988).
The design of idealized α/β‐barrels: Analysis of β‐sheet closure requirements
Authors: Lasters Ignace , Wodak Shoshana J. , Pio Fredric
Published: May 2005

The 8‐old parallel α/β‐barrel topology is encountered in proteins that display an impressive variety of functions, suggesting that this topology may be a rather nonspecific and stable folding motif. Consequently, this motif can be considered as an interesting framework to design novel proteins. It has been shown that the shape of the β‐sheet portion of the barrel can be approximated by a hyperboloid. This geometric object may therefore be used as a scaffold to construct an idealized eight‐standard β‐barrel. To facilitate the de novo design of such structures, a collection of modelling tools has been developed allowing secondary structure elements to be mapped onto the scaffold surface and rotation and translation operations to be performed about user defined axes while evaluating their contribution to the conformational energy of the system. These tools have been applied in a systematic study assessing the ϕ, ψ requirements to design symmetric eight standard β barrels with optimal hydrogen bonding between adjacent β‐strands. It is observed that: (a) the β‐sheet structure can be closed without introducing irregular stagger between β‐strands and (b) the region of ϕ, ψ dihedral angle space compatible with the formation of regular symmetric eight standard β‐barrels coincides with the ϕ, ψ region corresponding to average β‐strands in known protein structures, suggesting that barrel closure does not impose gross constraints on β‐strand geometry.
The characterization of recombinant mouse glandular kallikreins from E. coli
Authors: Blaber Michael , Isackson Paul J. , Bradshaw Ralph. A.
Published: May 2005

A system has been developed or the expression in E. coli of 12 of the 14 expressed mouse submandibular gland kallikreins as cassettes subcloned directly from cDNA. Using the epidermal growth factor binding protein (mGK‐9) and the γ‐subunit of nerve growth factor (nGK)‐3, as test cases, mature processed forms, obtained as functionally active proteins, as well as various precursor forms, were isolated. The expression system described allows rapid isolation of kallikrein protein from corresponding cDNA with yields of approximately 1.0 mg of purified protein from 10 g of initial cell paste. This expression system will facilitate structure/function studies of the mouse glandular kallikrein gene family and help elucidate the regions of the mature proteins responsible for the diverse catalytic behavior and growth factors interactions observed in this family of proteins.
Generation of potential structures for the G‐domain of chloroplast EF‐Tu using comparative molecular modeling
Authors: Lapadat Mary A. , Deerfield David W. , Pedersen Lee G. , Spremulli Linda L.
Published: May 2005

Comparative molecular modeling has been used to generate several possible structures for the G‐domain of chloroplast elongation factor Tu (EF‐Tuchl) based on the crystallographic data of the homologous E. coli protein. EF‐Tuchl contains a 10 amino acid insertion not present in the E. coli protein and this region has been modeled based on its predicted secondary structure. The insertion appears to lie on the surface of the protein. Its orientation could not be determined unequivocally but several likely structures for the nucleotide binding domain of EF‐Tuchl have been developed. The effects of the presence of water in the Mg2+ coordination sphere and of the protonation sate of the GDP ligand on the conformation of the guanine nucleotide binding site have been examined. Relative binding constants of several guanine nucleotide analogs for EF‐Tuchl have been obtained. The interactions between EF‐Tuchl and GDP predicted to be important by the models that have been developed are discussed in relation to the nucleotide binding properties of this factor and to the interactions proposed to be important in the binding of guanine nucleotides to related proteins.
Plastic adaptation toward mutations in proteins: Structural comparison of thymidylate synthases
Authors: Perry Kathy M. , Fauman Eric B. , Finer‐Moore Janet S. , Montfort William R. , Maley Gladys F. , Maley Frank , Stroud Robert M.
Published: May 2005

The structure of thymidylate synthase (TS) from Escherichia coli was solved from cubic crystals with a = 133 Å grown under reducing conditions at pH 7.0, and refined to R = 22% at 2.1 Å resolution. The structure is compared with that from Lactobacillus casei solved to R = 21% at 2.3 Å resolution. The structures are compared using a difference distance matrix, which identifies a common core of residues that retains the same relationship to one another in both species. After subtraction of the effects of a 50 amino acid insert present in Lactobacillus casei, differences in position of atoms correlate with temperature factors and with distance from the nearest substituted residue. The dependence of structural difference on thermal factor is parameterized and reflects both errors in coordinates that correlate with thermal factor, and the increased width of the energy well in which atoms of high thermal factor lie. The dependence of structural difference on distance from the nearest substitution also depends on thermal factors and shows an exponential dependence with half maximal effect at 3.0 Å from the substitution. This represents the plastic accommodation of the protein which is parameterized in terms of thermal B factor and distance from a mutational change.
The structure of rubredoxin from Desulfovibrio desulfuricans strain 27774 at 1.5 Å resolution
Authors: Stenkamp Ronald E. , Sieker Larry C. , Jensen Lyle H.
Published: May 2005

The structure of a small rubredoxin from the bacterium Desulfovibrio desulfuricans has been determined and refined at 1.5 Å resolution. The hairpin loop containing seven residues in other rubredoxins is missing in this 45 residue molecule, and once that fact was determined by amino acid sequencing studies, refinement progressed smoothly to an R value of 0.093 for all reflections from 5 to 1.5 Å resolution. Nearly all of the water molecules in the well‐ordered triclinic unit cell have been added to the crystallographic model. As in the other refined rubredoxin models, the Fe‐S4 complex is slightly distorted from ideal tetrahedral coordination.
Rate of β‐structure formation in polypeptides
Authors: Finkelstein A. V.
Published: May 2005

An explanation is suggested for why a marginally stable β‐structure folds extremely slowly; it is predicted that even a small increase in stability drastically accelerates β‐folding. According to the theory, this folding is a first‐order phase transition, and the rate‐limiting step is nucleation. The rate‐determining “nucleus” (transition state) is the smallest β‐sheet that is sufficiently large to provide an overall free energy reduction during subsequent folding. If the stability of the β‐structure is low, the nucleus is large and possesses a high free energy due to having a large perimeter. When the net stability of the final β‐structure increases (due to either an increase of the β‐sheet stability or a decrease in stability of the competing structures, e.g., α‐helices), the size and energy of a nucleus decrease and the rate of folding increases exponentially. This must result in a fast folding of polypeptides enriched by β‐forming residues (e.g., protein chains). The theory is developed for intramolecular β‐structure, but it can also explain the overall features of intermolecular β‐folding; it is applicable both to antiparallel and parallel β‐sheets. The difference in folding of β‐sheets, α‐helices, and proteins is discussed.
A workbench for multiple alignment construction and analysis
Authors: Schuler Gregory D. , Altschul Stephen F. , Lipman David J.
Published: May 2005

Multiple sequence alignment can be a useful technique for studying molecular evolution, as well as for analyzing relationships between structure or function and primary sequence. We have developed for this purpose an interactive program, MACAW (Multiple Alignment Construction and Analysis Workbench), that allows the user to construct multiple alignments by locating, analyzing, editing, and combining “blocks” of aligned sequence segments. MACAW incorporates several novel features. (1) Regions of local similarity are located by a new search algorithm that avoids many of the limitations of previous techniques. (2) The statistical significance of blocks of similarity is evaluted using a recently developed mathematical theory. (3) Candidate blocks may be evaluated for potential inclusion in a multiple alignment using a variety of visualization tools. (4) A user interface permits each blocks to be edited by moving its boundaries or by eliminating particular segments, and blocks may be linked to form a composite multiple alignment. No completely automatic program is likely to deal effectively with all the complexities of the multiple alignment problem; by combining a powerful similarity search algorithm with flexible editing, analysis and display tools, MACAW allows the alignment strategy to be tailored to the problem at hand.
Comparative modeling of mammalian aspartate transcarbamylase
Authors: Scully Joshua L. , Evans David R.
Published: May 2005

Mammalian aspartate transcarbamylase (ATCase) is part of a 243 kDa multidomain polypeptide, called CAD, that catalyzes the first three steps in de novo pyrimidine biosynthesis. The structural organization of the mammalian enzyme is very different from E. coli ATCase, a dodecameric, monofunctional molecule comprised of six copies of separate catalytic and regulatory chains. Nevertheless, sequence similarities and other properties suggested that the mammalian ATCase domain and the E. coli ATCase catalytic chain have the same tertiary fold. A model of mammalian ATCase was built using the X‐ray coordinates of the E. coli catalytic chain as a tertiary template. Five small insertions and deletions could be readily accommodated in the model structure. Following energy minimization the RMS difference in the α carbon positions of the mammalian and bacterial proteins was 0.93 Å. A comparison of the hydrophobic energies, surface accessibility index, and the distribution of hydrophilic and hydrophobic residues of the CAD ATCase structure with correctly and incorrectly folded proteins and with several X‐ray structures supported the validity of the model. The mammalian ATCase domain associates to form a compact globular trimer, a prerequisite for catalysis since the active site is comprised of residues from adjacent subunits. Interactions between the clearly defined aspartate and carbamyl phosphate subdomains of the monomer were largely preserved while there was appreciable remodeling of the trimeric interfaces. Several clusters of basic residues are located on the upper surface of the domain which account in part for the elevated isoelectric point (pI = 9.4) and may represent contact regions with other more acidic domains within the chimeric polypeptide. A long interdomain linker connects the monomer at its upper surface to the remainder of the polypeptide. The configuration of active site residues is virtually identical in the mammalian and bacterial enzymes. While the CAD ATCase domain can undergo the local conformational changes that accompany catalysis in the E. coli enzyme, the high activity, closed conformation is probably more stable in the mammalian enzyme.
The adaptability of the active site of trypanosomal triosephosphate isomerase as observed in the crystal structures of three different complexes
Authors: Noble Martin E. M. , Wierenga Rik K. , Lambeir Anne‐Marie , Opperdoes Fred R. , Thunnissen Andy‐Mark W. H. , Kalk Kor H. , Groendijk Hillie , Hol Wim G. J.
Published: May 2005

Crystals of triosephosphate isomerase from Trypanosoma brucei brucei have been used in binding studies with three competitive inhibitors of the enzyme's activity. Highly refined structures have been deduced for the complexes between trypanosomal triosephosphate isomerase and a substrate analogue (glycerol‐3‐phosphate to 2.2 Å), a transition state analogue (3‐phosphonopropionic acid to 2.6 Å), and a compound structurally related to both (3‐phosphoglycerate to 2.2 Å). The active site structures of these complexes were compared with each other, and with two previously determined structures of triosephosphate isomerase either free from inhibitor or complexed with sulfate. The comparison reveals three conformations available to the “flexible loop” near the active site of triosephosphate isomerase: open (no ligand), almost closed (sulfate), and fully closed (phosphate/phosphonate complexes). Also seen to be sensitive to the nature of the active site ligand is the catalytic residue Glu‐167. The side chain of this residue occupies one of two discrete conformations in each of the structures so far observed. A “swung out” conformation unsuitable for catalysis is observed when sulfate, 3‐phosphoglycerate, or no ligand is bound, while a “swung in” conformation ideal for catalysis is observed in the complexes with glycerol‐3‐phosphate or 3‐phosphonopropionate. The water structure of the active site is different in all five structures. The results are discussed with respect to the triosephosphate isomerase structure function relationship, and with respect to an on‐going drug design project aimed at the selective inhibition of glycolytic enzymes of T. brucei.
Free energy calculations on binding and catalysis by α‐lytic protease: The role of substrate size in the P1 pocket
Authors: Caldwell James W. , Agard David A. , Kollman Peter A.
Published: May 2005

We present free energy calculations using molecular dynamics on different substrates of α‐lytic protease in the gas phase, in solution, while forming a noncovalent Michaelis complex with the enzyme, and in a tetrahedral structure representing a transition state/intermediate for acylation by the enzyme. Various P1 substrates were studied, with P1 = Gly, Ala, Val, and Leu. In qualitative agreement with experiment, the enzyme was calculated to bind and catalyze most effectively substrates with P1 = Ala over those with P1 = Gly, Val or Leu. Also, the calculated relative solvation free energies of Gly → Ala and Ala → Val were in qualitative agreement with experimental values in corresponding model systems. However, the level of quantitative agreement with experiment achieved in our earlier study of relative binding and catalysis of native subtilisin and an Asn‐155 → Ala mutant was not achieved. We surmise that this is due to the greater difficulty in quantitatively simulating effects that are predominantly van der Waals and hydrophobic compared to those that are hydrogen bonding/electrostatic.
Gly‐238‐Ser substitution changes the substrate specificity of the SHV class A β‐lactamases
Authors: Lee Ki‐Young , Hopkins John D. , Syvanen Michael , O'Brien Thomas F.
Published: May 2005

The SHV‐type β‐lactamase SHV‐2A is related to SHV‐1 by a Gly‐238‐Ser replacement. Strains carrying SHV‐2A are resistant to the third generation cephems cefotaxime and ceftizoxime, whereas those that carry SHV‐1 are sensitive to these drugs. We present a kinetic analysis of a SHV‐1 and SHV‐2A enzymes, with the goal of gaining insight into the role of residue 238 in hydrolyzing cefotaxime and ceftizoxime. SHV‐2A shows altered kinetic properties for a number of other cephems that also have heterocyclic side chains at the amino position of the 7‐aminocephalosporanic acid nucleus (R1 side chain), including a significantly higher kcat/Km than does SHV‐1 for cephaloridine, cephalothin, and cefotiam. Two cephems with straight chain R1 substitutions, cephalosporin C and cephacetrile, are not hydrolyzed more efficiently by SHV‐2A. These results indicate that the Ser‐238‐Gly substitution increases the affinity toward cephems with a heterocyclic ring in the R1 side chain. In addition, the data for ampicillin and benzylpenicillin show that addition of a nitrogen to the second carbon of the R1 side chain of a penem results in a lower kcat/Km for SHV‐2A relative to SHV‐1. These data strongly suggest that the previously proposed hydrogen bond formation between Ser‐238 and the second carbon nitrogen of cefotaxime is not an important factor in hydrolysis by SHV‐2A. We propose that the Gly‐238 to Ser‐238 replacement in SHV‐2A has altered the hydrophobic pocket so that it can better accommodate cephems with bulky R1 side chains.
Protein‐protein recognition analyzed by docking simulation
Authors: Cherfils Jacqueline , Duquerroy Stéphane , Janin Joël
Published: May 2005

Antibody–lysozyme and protease–inhibitor complexes are reconstituted by docking lysozyme as a rigid body onto the combining site of the antibodies and the inhibitors onto the active site of the proteases. Simplified protein models with one sphere per residue are subjected to simulated annealing using a crude energy function where the attractive component is proportional to the interface area. The procedure finds clusters of orientations in which a steric fit between the two protein components is achieved over a large contact surface. With five out of six complexes, the native structure of the complexes determined by X‐ray crystallography is among those retained. Docked complexes are then subjected to conformational energy refinement with full atomic detail. With Fab HyHEL 5 and lysozyme, a native‐like complex has the lowest refined energy. It can also be retrieved when starting with the X‐ray structure of free lysozyme. However, some non‐native complexes cannot be rejected: they form large interfaces, have a large number of H‐bonds, and few unpaired polar groups. While these are necessary features of protein–protein recognition, they are not sufficient in determining specificity.
Functional implications of interleukin‐1β based on the three‐dimensional structure
Authors: Veerapandian B. , Gilliland Gary L. , Raag Reetta , Svensson Anders L. , Masui Yoshihiro , Hirai Yoshikatsu , Poulos Thomas L.
Published: May 2005

The molecular structure of interleukin‐1β, a hormone‐like cytokine with roles in several disease processes, has been determined at 2.0 Å resolution and refined to a crystallographic R‐factor of 0.19. The frame‐work of this molecule consists of 12 antiparallel β‐strands exhibiting pseudo‐3‐fold symmetry. Six of the strands make up a β‐barrel with polar residues concentrated at either end. Analysis of the three‐dimensional structure, together with results from site‐directed mutagenesis and biochemical and immunological studies, suggest that the core of the β‐barrel plays an important functional role. A large patch of charged residues on one end of the barrel is proposed as the binding surface with which IL‐1 interacts with its receptor.
Crystallization studies of glycosylated and unglycosylated human recombinant interleukin‐2
Authors: Stura Enrico A. , Chen Ping , Wilmot Carrie M. , Arevalo Jairo H. , Wilson Ian A.
Published: May 2005

Glycosylated interleukin‐2 (glyIL‐2) has been crystallized in two crystal forms, and unglycosylated interleukin‐2 (uIL‐2) has been crystallized in three forms. The glycosylated form of the human recombinant IL‐2 has been crystallized from 1.9 M ammonium sulfate, pH 6.5 to 7.0 in the hexagonal space group P6222 or its enantiomorph. The crystals diffract to 2.8 Å and contain two or three molecules per asymmetric unit. A second crystal form grows from 1.4 to 1.5 M ammonium sulfate in 0.2 M ammonium acetate, pH 5.0–5.5, as polycrystalline rosettes which are not suitable for even a preliminary crystallographic analysis. The uIL‐2 crystallizes from 1.0 to 1.7 M ammonium sulfate, 0.2 M ammonium acetate, pH 4.5–5.6 in the monoclinic space group P21, and less frequently in the orthorhombic space group P212121 from 2.5 M ammonium sulfate, pH 4.5 to 5.7. Cross‐seeding uIL‐2 with seeds from hexagonal crystals of glyIL‐2 promotes nucleation of trigonal crystals of unglycosylated IL‐2. These trigonal crystals belong to the space group P3121 or its enantiomorph, with similar cell dimensions to the glyIL‐2 hexagonal crystals.