bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural determinants of the conformations of medium‐sized loops in proteins
Authors: Tramontano Anna , Chothia Cyrus , Lesk Arthur M.
Published: May 2005

Loops are integral components of protein structures, providing links between elements of secondary structure, and in many cases contributing to catalytic and binding sites.

The conformations of short loops are now understood to depend primarily on their amino acid sequences. In contrast, the structural determinants of longer loops involve hydrogen‐bonding and packing interactions within the loop and with other parts of the protein. By searching solved protein structures for regions similar in main chain conformation to the antigen‐binding loops in immunoglobulins, we identified medium‐sized loops of similar structure in unrelated proteins, and compared the determinants of their conformations.

For loops that form compact substructures the major determinant of the conformation is the formation of hydrogen bonds to inward‐pointing main chain atoms. For oops that have more extended conformations, the major determinant of their structure is the packing of a particular residue or residues against the rest of the protein.

The following picture emerges: Medium‐sized lops of similar conformation are stabilized by similar interaction. The groups that interact with the loop have very similar spatial dispositions with respect to the loop. However, the residues that provide these interactions may arise from dissimilar parts of the protein: The conformation of the loop requires certain interactions that the protein may provide in a variety of ways.
A mutant T4 lysozyme (Val 131 → Ala) designed to increase thermostability by the reduction of strain within an α‐helix
Authors: Dao‐Pin S. , Baase W. A. , Matthews B. W.
Published: May 2005

An attempt has been made to identify residues in T4 phage lyoszyme that may have strained conformations and, by appropriate site‐directed replacements, to reduce this strain and thus increase the thermostability of the protein. Valine 131, within α‐helix 126–134, was identified as a potential candidate. Its side‐chain rotational as a potential candidate. Its side‐chain rotation angle, χ1, differs by approximately 18° from the low‐energy trans configuration. In addition, it is largely solvent exposed, yet is held in a rigid conformation. The mutant protein with Val 131 replaced by alanine temperature 0.9°C higher than that of wildtype lyoszyme at pH 2.8. As a control, The mutant Val 131 → Thr was also constructed and its melting temperature was found to be marginally lower than wild type. Higher‐resolution crystal structure determination of the mutant lysozymes show that their structure are virtually identical with that of wild‐type lyoszyme, except for the Val → Ala or Val → Thr replacement. Analysis of the different structures suggests that the design of the Val→Ala substitution was, in principle, successful, although the apparent gain in stability caused by reduction in strain is modest and is somewhat offset by the loss of hydrophobic interactions and by entrophic effects. The results also help to provide a structural retionalization that alanine has a higher helix propensity than valine or theronine.
Identification of protein folds: Matching hydrophobicity patterns of sequence sets with solvent accessibility patterns of known structures
Authors: Bowie James U. , Clarke Neil D. , Pabo Carl O. , Sauer Robert T.
Published: May 2005

Hydrophobic side chains often are buried in the interior of a protein, and evolutionarily related proteins usually maintain the hydrophobic character of buried position. In this paper we shown that a pattern of hydrophobicity values derived from a set of related protein sequences is well correlated with the linear pattern of side‐chain solvent accessibility values, calculated from a known protein structure representative of the sequences. In several cases, information from aligned sequences can be used to select the correct tertiary fold from a large database of protein structures.
HERA—A program to draw schematic diagrams of protein secondary structures
Authors: Hutchinson E. Gail , Thornton Janet M.
Published: May 2005

A program is described which generates hydrogen bonding diagrams of protein structures and optionally helical wheels and helical nets. The program can also be used simply to calculate the connectivities of β‐strands and to automatically extract simple structural motifs such as hairpins or Greek keys. The program greatly reduces the effort required to produce these diagrams and offers considerable flexibility in the information which can be represented. The usefulness of the program is illustrated by several examples including comparing homologous families, correlating protein structure with attributes of individual residues, and extracting all examples of the ψ‐loop motif from the Brookhaven Data Bank.
Three‐Dimensionnal structures of complexes of Lathyrus ochrus isolectin I with glucose and mannose: Fine specificity of the monosaccharide‐binding site
Authors: Bourne Yves , Roussel Alain , Frey Michel , Rougé Pierre , Fontecilla‐Camps Juan‐C , Cambillau Christian
Published: May 2005

The structure of the methyl‐α‐D‐mannopyranoside–LOL I complex has been solved by the molecular replacement method using the refined saccharide‐free LOL I coordinates as starting model. The methyl‐α‐D‐mannopyranoside–LOL I complex was refined by simulated annealing using the program X‐PLOR. The final R‐factor value is 0.182 [Fo > 1σ(Fo)]. The isostructural methyl‐α‐D‐glucopyranoside‐LOL I complex was refined by X‐Ray coupled energy minimization using the methyl‐α‐D‐mannopyranoside‐LOL I structure as a starting model to an R factor of 0.179 (all data). In both crystal forms, each dimer binds two molecules of sugar in pockets found near the calcium ions. The two saccharide moieties, which are in the C1 chair conformation, establish the same hydrogen bond pattern with the lectin. However, the van der Walls contacts are different between the O2, C2, C6, and O6 atoms of the two molecules and the backbone atoms of residues 208–211. Mannose, due to its axial C2 conformation, encloses the backbone atoms of the protein in a clamplike way. Van der Waals energy calculations suggest that this better complementarity of the mannoside molecule with the lectin could explain its higher affinity for isolectin I.
Design and synthesis of the pseudo‐EF hand in calbindin D9K: Effect of amino acid substitutions in the α‐helical regions
Authors: Tsuji Takashi , Kaiser Emil T.
Published: May 2005

A series of 37‐residue analogues of the pseudo‐EF hand in bovine calbindin D9K has been synthesized by the solid phase method. In the presence of calcium an α‐helical induction of up to 44% was observed for the peptide with the native sequence with a Kd for calcium binding of 0.35 mM. A number of amino acid substitutions have been carried out to study the packing of the two α‐helices based on the crystal structure of the entire protein. Three strategies were employed: (1) replacement of the Leu residues, which in the crystal structure do not contribute to the hydrophobic interaction between the two helices, by Gln or Ala in order to control the orientation of the helix packing, (2) stabilization of the individual helix by introducing a Glu−…︁Lys+ salt bridge or by changing the N‐terminal charge to compensate for the helix dipole moment, and (3) introduction of a disulfide bond between the two helices to help the packing of the helices.

The mutants with the substitution of (Leu‐30, Leu‐32) to (Gln‐30, Gln‐32), (Gln‐30, Ala‐32), and (Ala‐30, Ala‐32) designed based on the strategy 1 do not show any affinity for calcium and have low α‐helicity. The Leu‐30 to Lys‐30 mutant designed to form a salt bridge between the side chains of Glu‐26 and Lys‐30 has an apparent Kd for calcium of 6.8 mM. Kd of the N‐terminal acetylated and succinylated mutants are 0.41 and 0.45 mM, respectively, and no increase in the α‐helix content relative to that of the natural sequence peptide is observed. The disulfide containing mutants, namely Tyr‐13, Leu‐31 to Cys‐31 and Tyr‐13, Leu‐31 to Cys‐13, hCys‐31, show apparent Kd values of 0.93 and 2.1 mM, respectively. The former mutant shows the highest α‐helix content among the peptides studied in the presence and absence of calcium. While it is difficult to construct an isolated and rigid helix–loop–helix motif with peptides of this size, introduction of a disulfide bond proved to be effective for this purpose.
Weak correlation between predictive power of individual sequence patterns and overall prediction accuracy in proteins
Authors: Rooman Marianne J. , Wodak Shoshana J.
Published: May 2005

Patterns in amino acid properties (polar, hydrophobic, etc.) that characterize secondary structure motifs are derived from a database containing 75 protein structures, with the aim of circumventing the limitations due to data base size so as to increase structure prediction score. Many such sequence–structure associations with high intrinsic predictive power are found, which turn out to be correct 78% of the time when applied individually to proteins outside the learning set. Based on these associations, a prediction method is developed, which reaches the socre of 62% on the 3 states α‐helix, β‐strand, and loop, without using additional constraints. Though this score is quite good compared to that of other available prediction methods, it is much lower than could be expected from the high intrinsic predictive power of the associations used. The reasons underlying this surprising result, which indicate that prediction score and intrinsic predictive power are only weakly coupled, are discussed. It is also shown that the size of the present database still seriously limits prediction scores, even when property patterns are used, and that higher scores are expected in large databases. Clues are provided on the relative influence of neglecting spatial interactions on prediction efficiency, suggesting that, in sufficiently large databases, predicted secondary structures would correspond to those formed early in the folding process. This hypothesis is tested by confronting present predictions with available experimental data on early protein folding intermediates and on small peptides that adopt a relatively stable conformation in water. Although admittedly there are still too few such data, results suggest that the hypothesis might be well founded.
Crystallization of yeast orotidine 5′‐monophosphate decarboxylase complexed with 1‐(5′‐phospho‐β‐D‐ribofuranosyl) barbituric acid
Authors: Bell Juliette B. , Jones Mary Ellen , Carter Charles W.
Published: May 2005

Using an incomplete factorial experimental design, we have identified conditions for crystallization of yeast orotidine 5′‐monophosphate decarboxylase (ODCase) in an unliganded state and complexed separately to two inhibitors: 6‐azauridine 5′‐monophosphate (aza‐UMP) and 1‐(5′‐phospho‐β‐D‐ribofuranosyl) barbituric acid (BMP). Crystals of X‐ray diffraction quality have been obtained of yeast ODCase complexed with BMP, a putative transition state analog inhibitor (Ki = 8.8 × 10−12 M). ODCase:BMP complex crystals with a hexagonal rod habit were grown from a solution initially containing 12 mg/ml ODCase (205 μM dimer) plus 450 μM BMP by microdialysis at 4°C against a mother liquor which consisted of 0.1 M Na‐PIPES‐acetate (pH 6.4), 37.5 μM BMP, 5 mM mercaptoethanol, 1% polyethylene glycol 400, and 2.3 M ammonium sulfate. Crystals were analyzed using precession photography and were assigned to trigonal space group R32 with unit cell dimensions a = b = 115 Å, c = 385 Å. The crystal density is 1.245 g/cm3 indicating the presence of two ODCase:BMP complex dimers (118 kDa each) per asymmetric unit with a packing density of 2.08 Å3/Da and 41% solvent content. The morphological habit of crystals of the ODCase:BMP complex changed when the initial ammonium sulfate concentration was increased in 0.05 M steps from 2.3 to 2.45 M. All of these crystals diffracted to at least 3.0 Å resolution over a period of several weeks at room temperature and are isomorphous.
A metal‐mediated hydride shift mechanism for xylose isomerase based on the 1.6 Å Streptomycs rubiginosus structure with xylitol and D‐xylose
Authors: Whitlow Marc , Howard Andrew J. , Finzel Barry C. , Poulos Thomas L. , Winborne Evon , Gilliland Gary L.
Published: May 2005

The crystal structure of recombinant Streptomyces rubiginous D‐xylose isomerase (D‐xylose keto‐isomerase, EC 5.3.1.5) solved by the multiple isomorphous replacement technique has been refined to R = 0.16 at 1.64 Å resolution. As observed in an earlier study at 4.0 Å (Carrell et al., J. Biol. Chem. 259: 3230–3236, 1984), xylose isomerase is a tetramer composed of four identical subunits. The monomer consists of an eight‐stranded parallel β‐barrel surrounded by eight helices with an extended C‐terminal tail that provides extensive contacts with a neighboring monomer. The active site pocket is defined by an opening in the barrel whose entrance is lined with hydrophobic residues while the bottom of the pocket consists mainly of glutamate, aspartate, and histidine residues coordinated to two manganese ions.

The structures of the enzyme in the presence of MnCl2, the inhibitor xylitol, and the substrate D‐xylose in the presence and absence of MnCl2 have also been refined to R = 0.14 at 1.60 Å, R = 0.15 at 1.71 Å, R = 0.15 at 1.60 Å, and R = 0.14 at 1.60 Å, respectively. Both the ring oxygen of the cyclic α‐D‐xylose and its C1 hydroxyl are within hydrogen bonding distance of NE2 of His‐54 in the structure crystallized in the presence of D‐xylose. Both the inhibitor, xylitol, and the extended form of the substrate, D‐xylose, bind such that the C2 and C4 OH groups interact with one of the two divalent cations found in the active site and the C1 OH with the other cation. The remainder of the OH groups hydrogen bond with neighboring amino acid side chains.

A detailed mechanism for D‐xylose isomerase is proposed. Upon binding of cyclic α‐D‐xylose to xylose isomerase, His‐54 acts as the catalytic base in a ring opening reaction. The ring opening step is followed by binding of D‐xylose, in volving two divalent cations, in an extended conformation. The isomerization of D‐xylose to D‐xylulose involves a metal‐mediated 1,2‐hydride shift. The final step in the mechanism is a ring closure to produce α‐D‐xylulose. The ring closing is the reverse of the ring opening step.

This mechanism accounts for the majority of xylose iomerase's biochemical properties, in cluding (1) the lack of solvent exchange between the 2‐position of D‐xylose and the 1‐pro‐R position of D‐xylulose, (2) the chemical modification of histidine and lysine, (3) the pH vs. activity profile, and (4) the requirement for two divalent cations in the mechanism.
Electron redistribution on binding of a substrate to an enzyme: Folate and dihydrofolate reductase
Authors: Bajorath Jürgen , Kitson David H. , Fitzgerald George , Andzelm Jan , Kraut Joseph , Hagler Arnold T.
Published: May 2005

The migration of electron density of a substrate (folate) on binding to an enzyme (dihydrofolate reductase) is studied by a quantum‐mechanical method originally developed in solid state physics. A significant polarization of the substrate is induced by the enzyme, toward the transition state of the enzymatic reaction, at the same time giving rise to “electronic strain energy” in the substrate and enhanced protein–ligand interactions. The spatial arrangement of protein charges that induces the polarization is identified and found to be structurally conserved for bacterial and vertebrate dihydrofolate reductases.
Topological mirror images in protein structure computation: An underestimated problem
Authors: Pastore Annalisa , Atkinson R. Andrew , Saudek Vladimir , Williams Robert J. P.
Published: May 2005

When calculating three‐dimensional structures from NMR data, alternative solutions with very large RMS deviation can be obtained. Sometimes local or global inversions of the protein folding can be observed. We call these different solutions topological mirror images, as they keep the correct amino acid chirality. They are observed when the number of restraints is insufficient and represent different solutions from the same scalar information. Therefore they are common in small peptides where the NMR data are often limited and the secondary structure is not very well defined. They can also be observed in large molecules in regions of higher flexibility. In our experience the observation of topological mirror images is independent of the efficiency of sampling of the algorithm used. We present four examples of proteins with different size and folding. We also discuss ways to distinguish among the different solutions.
The crystal structure of the “open” and the “closed” conformation of the flexible loop of trypanosomal triosephosphate isomerase
Authors: Wierenga Rik K. , Noble Martin E. M. , Postma Johan P. M. , Groendijk Hillie , Kalk Kor H. , Hol Wim G. J. , Opperdoes Fred R.
Published: May 2005

Triosephosphate isomerase has an important loop near the active site which can exist in a “closed” and in an “open” conformation. Here we describe the structural properties of this “flexible” loop observed in two different structures of trypanosomal triosephosphate isomerase. Trypanosomal triosephosphate isomerase, crystallized in the presence of 2.4 M ammonium sulfate, packs as an asymmetric dimer of 54,000 Da in the crystallographic asymmetric unit. Due to different crystal contacts, peptide 167–180 (the flexible loop of subunit‐1) is an open conformation, whereas in subunit‐2, this peptide (residues 467–480) is in a closed conformation. In the closed conformation, a hydrogen bond exists between the tip of the loop and a well‐defined sulfate ion which is bound to the active site of subunit‐2. Such an active site sulfate is not present in subunit‐1 due to crystal contacts. When the native (2.4 M ammonium sulfate) crystals are transferred to a sulfate‐free mother liquor, the flexible loop of subunit‐2 adopts the open conformation. From a closed starting model, this open conformation was discovered through molecular dynamics refinement without manual intervention, despite involving Cα shifts of up to 7 Å. The tip of the loop, residues 472, 473, 474, and 475, moves as a rigid body. Our analysis shows that in this crystal form the flexible loop of subunit‐2 faces a solvent channel. Therefore the open and the closed conformations of this flexible loop are virtually unaffected by crystal contacts. The actual observed conformation depends only on the absence or presence of a suitable ligand in the active site.
Relaxation data in NMR structure determination: Model calculations for the lysozyme–Gd3+ complex
Authors: Sutcliffe Michael J. , Dobson Christopher M.
Published: May 2005

The effect of including paramagnetic relaxation data as additional restraints in the determination of protein tertiary structures from NMR data has been explored by a systematic series of model calculations. The system used for testing the method was the 2.0 Å resolution tetragonal crystal structure of hen egg white lysozyme (129 amino acid residues) and structures were generated using a version of the hybrid “distance geometry–dynamic simulated annealing” procedure. A limited set of 769 NOEs was used as restraints in all the calculations; the strengths of these were categorized into three classes on the basis of distances observed in the crystal structure. The values of 50 ϕ angles were also restrained on the basis of amide–alpha coupling constants calculated from the X‐ray structure. Five sets of 12 structures were determined using differing sets of paramagnetic relaxation data as restraints additional to those involving the NOE and coupling constant data. The paramagnetic relaxation data were modeled on the basis of the distances of defined protons from the crystallographic binding site of Gd3+ in lysozyme. Analysis of the results showed that the relaxation data significantly improved the correspondence between the set of generated structures and the crystal structure, and that the more well defined the relaxation data, the more significant the improvement in the quality of the structures. The results suggest that the inclusion of paramagnetic relaxation restraints could be of significant value for the experimental determination of protein structures from NMR data.
Modeling conformational change in macromolecules as an elastic deformation
Authors: Andrews Lawrence C. , Harrison Robert W.
Published: May 2005

Macromolecules are elastic bodies. Atomic strucutres are available for nucleic acids and proteins in two or more different conformations. It is a common practice to compare two structures by finding the best rigid body superposition of the molecules. This ignores possible deformations. There is useful information in the deviations from the rigid body superposition. If the deviations are considered to be elastic deformations of a common structure than it is possible to extract this information. Results are shown for comparisons of deoxyhemoglobin versus carbonmonoxyhemoglobin and for two different conformations of catabolite gene activator protein.
Secondary structure‐based profiles: Use of structure‐conserving scoring tables in searching protein sequence databases for structural similarities
Authors: Lüthy Roland , McLachlan Andrew D. , Eisenberg David
Published: May 2005

The profile method, for detecting distantly related proteins by sequence comparison, has been extended to incorporate secondary structure information from know X‐ray structures. The sequence of a known structure is aligned to sequences of other members of a given folding class. From the known structure, the secondary structure (α‐helix, β‐strand or “other”) is assigned to each position of the aligned sequences. As in the standard profile method,1 a position‐dependent scoring table, termed a profile, is calculated from the aligned sequences. However, rather than using the standard Dayhoff mutation table in calculating the profile, we use distinct amino acid mutation tables for residues in α‐helices, β‐strands or other secondary structures to calculate the profile. In addition, we also distinguish between internal and external residues. With this new secondary structure‐based profile method, we created a profile for eight‐stranded, antiparallel β barrels of the insecticyanin folding class. It is based on the sequences of retinol‐binding protein, insecticyanin and β‐lactoglobulin. Scanning the sequence database with this profile, it was possible to detect the sequence of avidin. The structure of streptavidin is known, and it appears to be distantly related to the antiparallel β barrels. Also detected is the sequence of complement component C8, which we therefore predict to be a member of this folding class.
Variation of folded polypeptide surface area with probe size
Authors: Islam Suhail A. , Weaver David L.
Published: May 2005

Three types of polypeptide surface area (contact, accessible, and molecular) have been studied as a function of the radius of a probe sphere used to map the surface. The surfaces are: (1) three α‐helices, the H‐helix of myoglobin, the E‐helix of leghemoglobin, and an artificial polyalanine helix, each with 26 residues; (2) two globins, myoglobin and leghemoglobin, each with 153 residues: and (3) a two center model system for which the three types of surface area have been calculated analytically. The two globin helices have almost identical surface areas as a function of probe size as do the two globins. The polyalanine helix surface area is smaller but similar in shape to the globin helix areas. All three helix contact areas tend to the same limit as the probe size increases, and the globin contact areas behave similarly. Fractal dimensions were calculated for the helix and globin contact and molecular surfaces. All fractal dimensions showed strong dependence on probe size. The contact fractal dimension peaks at larger values for both the helices and globins. Most residues do not make contact with large probes (15 Å).
Molecular dynamics simulations of the cytochrome c3–rubredoxin complex from Desulfovibrio vulgaris
Authors: Stewart David E. , Wampler John E.
Published: May 2005

Molecular dynamics simulations have been carried out on the complex formed between the tetraheme cytochrome c3 and the iron protein rubredoxin from the sulfate‐reducing bacterium Desulfovibrio vulgaris. These simulations were performed both with explicit solvent water molecules included, and without solvent molecules using a distance‐dependent dielectric constant to approximate the screening effects of solvent. The results of both simulations are strikingly different, indicating that the representation of environmental effects is important in such simulations. For example, a striking adaptation of the two proteins seen in the nonsolvated simulation is not seen when explicit solvent water is included; in fact, the complex appears to become weaker in the solvated simulation. Nonetheless, the iron–iron distance decreases more significantly in the solvated simulation than in the nonsolvated simulation. It was found that in both cases molecular dynamics optimized the structures further than energy minimization alone.
A 175‐psec molecular dynamics simulation of camphor‐bound cytochrome P‐450cam
Authors: Paulsen Mark D. , Ornstein Rick L.
Published: May 2005

The structure and internal motions of cytochrome P‐450cam, a monooxygenase heme enzyme with 414 amino acid residues, with camphor bound at the active site have been evaluated on the basis of a 175‐psec molecular dynamics simulation carried out at 300 K. All hydrogen atoms were explicitly modeled, and 204 crystallographic waters were included in the simulation. Based on an analysis of the time course of the trajectory versus potential energy, root mean square deviation, radius of gyration, and hydrogen bonding, the simulation was judged to be stable and representative of the average experimental structure. The averaged structural properties of the enzyme were evaluated from the final 135 psec of the simulation. The average atomic displacement from the X‐ray structure was 1.39 Å for all heavy atoms and 1.17 Å for just C‐α atoms. The average root‐mean‐square (rms) fluctuations of all heavy atoms and backbone atoms were 0.42 and 0.37 Å, respectively. The computed rms fluctuations were in reasonable agreement with the experimentally determined temperature factors. All 13 segments of α‐helix and 5 segments of β‐sheet were well preserved with the exception of the N‐terminal half of halix F which alternated between an α‐helix and a 310‐helix. In addition there were in general only small variations in the relative orientation of adjacent α‐helices. The rms fluctuations of the backbone dihedral angles in the secondary structure elements were almost uniformly smaller, with the fluctuation in α‐helices and β‐sheets, 31 and 10% less, respectively, than those in nonsecondary structure regions. The reported crystal structure contains kinks in both helices C and I. In the simulation, both of these regions showed high mobility and large deviations from their starting positions. Since the kink in the I helix is at the oxygen binding site, these motions may have mechanistic implications.
Structure–function relationships of the hematopoietic growth factors
Authors: Kaushansky Kenneth
Published: May 2005

The hematopoietic growth factors are a family of glycoproteins involved in the production of blood cells from their bone marrow precursors and in the activation of mature blood cells. Much has been learned about the structural features of these molecules responsible for their characteristic biological activities. Most studies have been based upon mutagenesis strategies of intact polypeptides and on epitope mapping of informative monoclonal antibodies to the growth factors. A more limited amount of physical data is available. This review will summarize these findings, highlight the growing body of evidence suggesting that many of these proteins share common evolutionary origins and structural elements, and hopefully point to the directions being taken for further investigations of these scientifically informative and clinically useful group of proteins.
CLIX: A search algorithm for finding novel ligands capable of binding proteins of known three‐dimensional structure
Authors: Lawrence Michael C. , Davis Paul C.
Published: May 2005

A computer algorithm, CLIX, capable of searching a crystallographic database of small molecules for candidates which have both steric and chemical likelihood of binding a protein of known three‐dimensional structure is presented. The algorithm is a significant advance over previous strategies which consider solely steric or chemical requirements for binding. The algorithm is shown to be capable of predicting the correct binding geometry of sialic acid to a mutant influenzavirus hemagglutinin and of proposing a number of potential new ligands to this protein.
Effects of eglin‐c binding to thermitase: Three‐dimensional structure comparison of native thermitase and thermitase eglin‐c complexes
Authors: Gros Piet , Teplyakov Alex V. , Hol Wim G. J.
Published: May 2005

Thermitase is a thermostable member of the subtilisin family of serine proteases. Four independently determined crystal structures of the enzyme are compared in this study: a high resolution native one and three medium resolution complexes of thermitase with eglin‐c, grown from three different calcium concentrations. It appeared that the B‐factors of the thermitase eglin complex obtained at 100 mM CaCl2 and elucidated at 2.0 Å resolution are remarkably similar to those of the 1.4 Å native structure: the main chain atoms have an rms difference of only 2.3 Å2; for all atoms this difference is 4.6 Å2. The rms positional differences between these two structures of thermitase are 0.31 Å for the main chain atoms and 0.58 Å for all atoms. There results show that not only atomic positions but also temperature factors can agree well in X‐ray structures determined entirely independently by procedures which differ in virtually every possible technical aspect.

A detailed comparison focussed on the effects of eglin binding on the structure of thermitase. Thermitase can be considered as consisting of (1) a central core of 94 residues, plus (2) four segments of 72 residues in total which shift as rigid bodies with respect to the core, plus (3) the remaining 113 residues which show small changes but, however, cannot be described as rigid bodies. The central cores of native thermitase and the 100 mM CaCl2 thermitase:eglin complex have an rms deviation of 0.13 Å for 376 main chain atoms. One of the segments, formed by loops of the strong calcium binding site, shows differences up to 1.0 Å in Cα positions. These are probably due to crystal packing effects.

The three other segments, comprising 51 residues, are affected conformational changes upon eglin binding so that the P1 to P3 binding pockets of thermitase broaden by 0.4 to 0.7 Å. The residues involved in these changes correspond with residues which change position upon inhibitor binding in other subtilisins. This suggests that an induced fit mechanism is operational during substrate recognition by subtilisins.
Redesigning the DNA‐binding specificity of a zinc finger protein: A data base‐guided approach
Authors: Desjarlais John R. , Berg Jeremy M.
Published: May 2005

A peptide corresponding to the three zinc finger domains of the human transcription factor Sp1 has been expressed and found to bind a consensus Sp1 binding site with the sequence 5′‐GGGGCGGGG‐3′. Examination of the amino acid distributions within a large zinc finger sequence data base and chemical arguments suggested that a particular Arg to Gln sequence change might convert binding specificity to 5′‐GGGGCAGGG‐3′. Experimental tests of this hypothesis revealed that such a change could be induced only when two other sequence changes, deduced from examination of sequence correlations, were made as well. These results provide the most direct information to date about how zinc finger proteins might recognize adenine‐containing binding sites and bear on the existence and nature of any code between zinc finger protein and binding site sequences.
The high‐resolution crystal structure of porcine pepsinogen
Authors: Hartsuck Jean A. , Koelsch Gerald , Remington S. James
Published: May 2005

The structure of porcine pepsinogen at pH 6.1 has been refined to an R‐factor of 0.173 for data extending to 1.65 Å. The final model contains 180 solvent molecules and lacks density for residues 157–161. The structure of this aspartic proteinase zymogen possesses many of the characteristics of pepsin, the mature enzyme. The secondary structure of the zymogen consists predominantly of β‐sheet, with an approximate 2‐fold axis of symmetry. The activation peptide packs into the active site cleft, and the N‐terminus (IP–9P) occupies the position of the mature N‐terminus (1–9). Thus changes upon activation include excision of the activation peptide and proper relocation of the mature N‐terminus. The activation peptide or residues of the displaced mature N‐terminus make specific interactions with the substrate binding subsites. The active site of pepsinogen is intact; thus the lack of activity of pepsinogen is not due to a deformation of the active site. Nine ion pairs in pepsinogen may be important in the advent of activation and involve the activation peptide or regions of the mature N‐terminus which are relocated in the mature enzyme. The activation peptide–pepsin junction, 44P‐1, is characterized by high thermal parameters and weak density, indicating a flexible structure which would be accessible to cleavage. Pepsinogen is an appropriate model for the structures of other zymogens in the aspartic proteinase family. © 1992 Wiley‐Liss, Inc.
Characterization of HIV‐1 p24 self‐association using analytical affinity chromatography
Authors: Rosé Sergio , Hensley Preston , O'Shannessy Daniel J. , Culp Jeffrey , Debouck Christine , Chaiken Irwin
Published: May 2005

Analytical affinity chromatography (AAC) was used to detect and quantitate the self‐association of p24gag, the major structural capsid protein of human immunodeficiency virus (HIV‐1). p24gag was immobilized on a hydrophilic polymer (methacrylate) chromatographic support. The resulting affinity column was able to interact with soluble p24, as judged by the chromatographic retardation of the soluble protein upon isocratic elution undernonchaotropic binding conditions. The variation of elution volume with soluble protein concentration fit to a monomer–dimer model for self‐association. The soluble p24–immobilized p24 association process was observed using both frontal and zonal elution AAC at varying pH values; the dissociation constant was 3–4 × 10−5 M at pH 7. That p24 monomer associates to dimers was determined in solution using analytical ultracentrifugation. The solution Kd was 1.3 × 10−5 M at pH 7. AAC in the zonal elution mode provides a simple and rapid means to screen for other HIV‐1 macromolecules that may interact with p24 as well as for modulators, including antagonists, of HIV p24 protein assembly. © 1992 Wiley‐Liss, Inc.
Modeling microdomains: The surface area of globin helices
Authors: Weaver David L.
Published: May 2005

The accessible surface areas of 53 high‐resolution globin helices are correlated with molecular weight. The linear fit is assessed for satistical accuracy using a boot‐strap analysis, and by comparison to the areas of 13 ideal polyalanine α‐helices. The accessible area of the unfolded helices is compared with the folded values before helix‐helix packing. An analytical physical model is presented to explain the correlation, and to provide an analytical value for the surface area parameter in the diffusion‐collision model of protein folding. © 1992 Wiley‐Liss, Inc.
Folding in vitro of bovine pancreatic trypsin inhibitor in the presence of proteins of the endoplasmic reticulum
Authors: Zapun André , Creighton Thomas E. , Rowling Pamela J. E. , Freedman Robert B.
Published: May 2005

The rate of folding and disulfide bond formation in reduced BPTI were measured in vitro in the presence and absence of total protein from the endoplasmic reticulum. The rates were increased substantially by the endoplasmic reticulum proteins, but only to the extent expected from the known content and activity of protein–disulfide–isomerase. No effects of added ATP or Ca2+ were observed, even though protein–disulfide–isomerase blinds Ca2+ tightly. © 1992 Wiley‐Liss, Inc.
A structural model for human dihydrolipoamide dehydrogenase
Authors: Jentoft Joyce E. , Shoham Menachem , Hurst Darren , Patel Mulchand S.
Published: May 2005

The hypothesis that dihydrolipoamide dehydrogenases (E3s) have tertiary structures very similar to that of human glutathione reductase (GR) was tested in detail by three separate criteria: (1) by analyzing each putative secondary structural element for conservation of appropriate polar/nonpolar regions, (2) by detailed comparison of putative active site residues in E3s with their authentic counterparts in human GR, and (3) by comparison of residues at the putative dimeric interface of the E3s with the authentic residues in GR. All three criteria are satisfied in a convincing way for the 7 E3s that were considered, supporting the conclusion that the structural scaffolding and the overall tertiary structure (which determines the location of functional sites and residues) are remarkably similar for the E3s and for GR. These analyses together with the crystal structures of human erythrocyte GR formed the basis for construction of a molecular model for human E3. The cofactor FAD and the substrakes NAD and lipoic acid were also included in the model. Unexpectedly, the surface residues in the cleft that holds the lipoamide were found to be highly charged and predominantly acidic, allowing us to predict that the region around the lipoamide in the sub‐unit should be basic in nature. The molecular model can be tested by site‐directed mutagenesis of residues predicted to be in the dihydrolipoamide acetyltransferase subunit binding cleft. © 1992 Wiley‐Liss, Inc.
The role of tyrosine 67 in the cytochrome c heme crevice structure studied by semisynthesis
Authors: Frauenhoff Mary M. , Scott Robert A.
Published: May 2005

Tyr‐67 of mitochondrial cytochrome c is thought to be involved in important hydrogen bonding interactions in the hydrophobic heme pocket of the protein (Takano, T., Dickerson, R.E. (1981) J. Mol. Biol. 153:95–115). The role of this highly conserved residue in heme pocket stability was studied by comparing properties of semisynthetic (Phe‐67) and (p‐F‐Phe‐67) analogs with those of native cytochrome c and a “control” analog, (Hse‐65)cytochrome c. The (Phe‐67) and (p‐F‐Phe‐67) analogs have well‐developed 695‐nm visible absorption bands and are active in a cytochrome c oxidase assay. The reduction potentials of both analogs are lower than the native protein by approximately 50 mV. Although both analogs bind imidazole with higher affinity than the native protein, only the (p‐F‐Phe‐67) analog has a 3‐ to 5‐fold lower binding constant for cyanide. Only the (Phe‐67) analog was significantly more stable toward alkaline isomerization. These results are not consistent with stabilization of the native protein heme pocket via hydrogen bonding of Tyr‐67 to Met‐80. An alternative steric role for Tyr‐67 is proposed in which the residue controls the heme reduction potential by limiting the number of internal H2O molecules in the heme pocket. © 1992 Wiley‐Liss, Inc.
Selection of a representative set of structures from brookhaven protein data bank
Authors: Boberg Jorma , Salakoski Tapio , Vihinen Mauno
Published: May 2005

Reliable structural and statistical analyses of three dimensional protein structures should be based on unbiased data. The Protein Data Bank is highly redundant, containing several entries for identical or very similar sequences. A technique was developed for clustering the known structures based on their sequences and contents of α‐and β‐structures. First, sequences were aligned pairwise. A representative sample of sequences was then obtained by grouping similar sequences together, and selecting a typical representative from each group. The similarity significance threshold needed in the clustering method was found by analyzing similarities of random sequences. Because three dimensional structures for proteins of same structural class are generally more conserved than their sequences, the proteins were clustered also according to their contents of secondary structural elements. The results of these clusterings indicate conservation of α‐and β‐structures even when sequence similarity is relatively low.

An unbiased sample of 103 high resolution structures, representing a wide variety of proteins, was chosen based on the suggestions made by the clustering algorithm. The proteins were divided into structural classes according to their contents and ratios of secondary structural elements. Previous classifications have suffered from subjectice view of secondary structures, whereas here the classification was based on backbone geometry. The concise view lead to reclassification of some structures. The representative set of structures facilitates unbiased analyses of relationships between protein sequence, function, and structure as well as of structural characteristics. © 1992 Wiley‐Liss, Inc.
Effect of polyethylene glycol‐400 at low concentrations on long‐term growth of muscle phosphoglucomutase crystals from concentrated salt solutions
Authors: Ray William J.
Published: May 2005

Although rabbit muscle phosphoglucomutase occasionally deposits tetragonal crystals from solutions of ammonium sulfate at about 47% of saturation, low concentrations of polyethylene glycol‐400 (PEG), 1 to 4.5% w/v, must be included to sustain crystal growth. A comparison of long‐term growth rates for macroscopic crystals in the presence and absence of added PEG suggests that at high salt concentration this cosolute exerts its primary effect on disordered protein aggregates, either in the external medium or at the surface of the crystal, and thereby allows the growth of much larger crystals. Since the observed effects may arise from a PEG‐induced increase in the “solubility” of the aggregate that exceeds the induced increase in solubility of the crystalline phase under these conditions, the physical basis for a cosolute‐induced increase in solubility in the presence of a precipitant is considered. The applicability of such a rationale to the present system is supported by an assessment of the relative effects of polyeth‐yiene glycol and β‐octylglucoside on amorphous, salt‐induced precipitates of phosphoglucomutase. PEG also produces what appears to be a differential effect on nucleation efficiency and crystal growth rate. Thus, seed crystals cannot be enlarged at a significant rate at high salt concentration without producing showers of extraneous nucleation centers when the concentration of added PEG is 3% or less. But PEG concentrations of 4.5% essentially eliminate the showering problem, ostensibly by increasing the supersaturation required for nucleation to a greater extent than that required for crystal growth. The same type of effect is observed during de novo growth. Again a solubility‐based mechanism is posed. Hysteretic effects related to properties of amorphous aggregates of the protein also are described. © 1992 Wiley‐Liss, Inc.
Mutagenic dissection of hemoglobin cooperativity: Effects of amino acidalteration on subunit assembly of oxy and deoxy tetramers
Authors: Turner George J. , Galacteros Frederic , Doyle Michael L. , Hedlund Bo , Pettigrew Donald W. , Turner Benjamin W. , Smith Francine R. , Moo‐Penn Winston , Rucknagel Donald L. , Ackers Gary K.
Published: May 2005

Free energies of oxygen‐linked subunit assembly and cooperative interaction have been determined for 34 molecular species of human hemoglobin, which differ by amino acid alterations as a result of mutation or chemical modification at specific sites. These studies required the development of extensions to our earlier methodology. In combination with previous results they comprise a data base of 60 hemoglobin species, characterized under the same conditions. The data base was analyzed in terms of the five following issues. (1) Range and sensitivity to site modifications. Deoxy tetramers showed greater average energetic response to structural modifications than the oxy species, but the ranges are similar for the two ligation forms. (2) Structural localization of cooperative free energy. Difference free energies of dimer‐tetramer assembly (oxy minus deoxy) yielded ΔGc for each hemoglobin, i.e., thefree energy used for modulation of oxygen affinity over all four binding steps. A structure‐energy map constructed from these results shows that the α1 β2 interface is a unique structural location of the noncovalent bonding interactions that are energetically coupled to cooperativity. (3) Relationship of cooperativity to intrinsic binding. Oxygen binding energetics for dissociated dimers of mutants strongly indicates that cooperativity and intrinsic binding are completely decoupled by tetramer to dimer dissociation. (4) Additivity, site‐site coupling and adventitious perturbations. All these are exhibited by individual‐site modifications of this study. Large nonadditivity may be correlated with global (quaternary) structure change.(5) Residue position vs. chemical nature. Functional response is solely dictated by structural location for a subset of the sites, but varies with side‐chain type at other sites. The current data base provides a unique framework for further analyses and modeling of fundamental issues in the structural chemistry of proteins and allosteric mechanisms. © 1992 Wiley‐Liss, Inc.
Limits on α‐helix prediction with neural network models
Authors: Hayward S. , Collins J. F.
Published: May 2005

Using a backpropagation neural network model we have found a limit for secondary structure prediction from local sequence. By including only sequences from whole α‐helix and non‐α‐helixstructures in our training and test sets—sequences spanning boundaries between these two structures were excluded—it was possible to investigate directly the relationship between sequence and structure for α‐helix. A group of non‐α‐helix sequences, that was disrupting overall prediction success, was indistinguishable to the network from α‐helix sequences. These sequences were found to occur at regions adjacent to the termini of α‐helices with statistical significance, suggesting that potentially longer α‐helices are disrupted by global constraints. Some of these regions spanned more than 20 residues. On these whole structure sequences, 10 residues in length, a comparatively high prediction success of 78% with a correlation coefficient of 0.52 was achieved. In addition, the structure of the input space, the distribution of β‐sheet in this space, and the effect of segment length were also investigated. © 1992 Wiley‐Liss, Inc.
Thermodynamic integration calculations of binding free energy difference for Gly‐169 mutation in subtilisin BPN′
Authors: Wang Cun Xin , Shi Yun Yu , Zhou Feng , Wang Lu
Published: May 2005

The binding free energy difference for the Gly‐169 → Ala‐169 (G169A) mutation in subtilisin BPN′ complexed with a tripeptide substrate analogue is explored using the thermodynamic integration approach. The structure of the mutant enzyme–substrate complex obtained from free energy simulation is in good agreement with experimental X‐ray refinement. The near perfect reversibility is obtained in the present work for ensuring the correctness of the free energy calculations. The results of the binding free energy difference are close to similar experimental data. © 1993 Wiley‐Liss, Inc.