bluuebunny/crossref_metadata_2025 huggingface dataset:
Metal ion stabilization of the conformation of a recombinant 19‐kDa catalytic fragment of human fibroblast collagenase
Authors: Lowry Cynthia L. , McGeehan Gerard , Le Vine Harry
Published: May 2005

A recombinant 19‐kDa human fibroblast collagenase catalytic fragment modeled on a naturally occurring proteolytic product was purified from E. coli inclusion bodies. Following renaturation in the presence of zinc and calcium, the fragment demonstrated catalytic activity with the same primary sequence specificity against small synthetic substrates as the full‐length collagenase. Unlike the parent enzyme, it rapidly cleaved casein and gelatin but not native type I collagen. Intrinsic fluorescence of the three tryptophan residues was used to monitor the conformational state of the enzyme, which underwent a 24‐nm red shift in emission upon denaturation accompanied by quenching of the fluorescence and loss of catalytic activity. Low concentrations of denaturant unfolded the fragment while the full‐length enzyme displayed a shallow extended denaturation curve. Calcium remarkably stabilized the 19‐kDa fragment, zinc less so, while together they were synergistically stabilizing. Among divalent cations, calcium was the most effective stabilizer, EC50 ∼60 μM, and similar amounts were required for substrate hydrolysis. Catalytic activity was more sensitive to denaturation than was tryptophan fluorescence. Least sensitive was the polypeptide backbone secondary structure assessed by CD. These observations suggest that the folding of the 19‐kDa collagenase fragment is a multistep process stabilized by calcium.
Characterization of a putative calcium‐binding site in tobacco mosaic virus
Authors: Pattanayek Rekha , Elrod Monicia , Stubbs Gerald
Published: May 2005

Lead has been used as a substitute for calcium binding to tobacco mosaic virus (TMV). The high atomic number of lead has allowed us to use difference maps from X‐ray fiber diffraction data to characterize a calcium‐binding site in the virus. The metal ligands are slightly different from those previously believed to bind calcium to TMV, although the binding site is very close to one previously described. Two acetate groups are also bound to the lead atom. There is no significant backbone conformational change in the protein as a result of metal binding; the binding is accomplished by means of relatively small movements in amino acid side chains.
A structural assessment of the apo[a] protein of human lipoprotein[a]
Authors: Guevara Juan , Knapp Roger D. , Honda Sandra , Northup S. Robert , Morrisett Joel D.
Published: May 2005

Apolipoprotein[a], the highly glycosylated, hydrophilic apoprotein of lipoprotein[a] (Lp[a]), is generally considered to be a multimeric homologue of plasminogen, and to exhibit atherogenic/thrombogenic properties. The cDNA‐inferred amino acid sequence of apo[a] indicates that apo[a], like plasminogen and some zymogens, is composed of a kringle domain and a serine protease domain. To gain insight into possible positive functions of Lp[a], we have examined the apo[a] primary structure by comparing its sequence with those of other proteins involved in coagulation and fibrinolysis, and its secondary structure by using a combination of structure prediction algorithms. The kringle domain encompasses 11 distinct types of repeating units, 9 of which contain 114 residues. These units, called kringles, are similar but not identical to each other or to PGK4. Each apo[a] kringle type was compared with kringles which have been shown to bind lysine and fibrin, and with bovine prothrombin kringle 1. Apo[a] kringles are linked by serine/threonine‐ and proline‐rich stretches similar to regions in immunoglobulins, adhesion molecules, glycoprotein Ib‐α subunit, and kininogen. In comparing the protease domains of apo[a] and plasmin, apo[a] contains a region between positions 4470 and 4492 where 8 substitutions, 9 deletions, and 1 insertion are apparent. Our analysis suggests that apo[a] kringle‐type 10 has a high probability of binding to lysine in the same way as PGK4. In the only human apo[a] polymorph sequenced to date, position 4308 is occupied by serine, whereas the homologous position in plasmin is occupied by arginine and is an important site for proteolytic cleavage and activation. An alternative site for the proteolytic activation of human apo[a] is proposed.
Functional significance of conserved amino acid residues
Authors: Poteete Anthony R. , Rennell Dale , Bouvier Suzanne E.
Published: May 2005

A systematic study of single amino acid substitutions in bacteriophage T4 lysozyme permitted a test of the concept that conserved amino acid residues are more functionally important than nonconserved residues. Substitutions of amino acid residues that are conserved among five bacteriophage‐encoded lysozymes were found to lead more frequently to loss of function than substitutions of nonconserved residues. Of 163 residues tested, only 74 (45%) are sensitive to at least one substitution; however, all 14 residues that are fully conserved are sensitive to substitutions. © 1992 Wiley‐Liss, Inc.
Monte Carlo docking of oligopeptides to proteins
Authors: Caflisch Amedeo , Niederer Peter , Anliker Max
Published: May 2005

A new two‐step procedure has been developed for the docking of flexible oligopeptide chains of unkown conformation to static proteins ofknown structure. In the first step positions and conformations are sampled and the association energy, minimized starting from an approximate preselected docking position. The resulting conformations are further optimized in the second step by a Metropolis Monte Carlo minimization, which optimizes each of these structures. The method has been tested on the HIV‐1 aspartic proteinase complex with an inhibitor, whose crystallographic structure is known at 2.3 Å resolution. Furthermore, the application of this method to the docking of the hendecapeptide 58–68 of the influenza A virus matrix protein to the HLA‐A2 molecule produced results which are in agreement with experimental observations in identifying side chains critical for T cell recognition and residues responsible of MHC protein binding. © 1992 Wiley‐Liss, Inc.
Molecular dynamics characterization of the active cavity of carboxypeptidase A and some of its inhibitor adducts
Authors: Banci Lucia , Schröder Stefan , Kollman Peter A.
Published: May 2005

Molecular dynamics (MD) calculations have been performed on carboxypeptidase A and on its adducts with inhibitors, such as d‐phenylalanine (dPhe) and acetate. The catalytically essential zinc ion present in the protein was explicitly included in all the simulations. The simulation was carried out over a sphere of 15 Å centered on the zinc ion. The crystallographic water molecules were explicitly taken into account; then the protein was solvated with a 18 Å sphere of water molecules. MD calculations were carried out for 45–60 ps. There is no large deviation from the available X‐ray structures of native and the dPhe adduct for the MD stuctures. Average MD structures were calculated starting from the X‐ray structure of the dPhe adduct, and, from a structure obtained by docking the inhibitor in the native structure. Comparison between these two structures and with that of the native protein shows that some of the key variations produced by inhibitor binding are reproduced by MD calculations. Addition of acetate induces structural changes relevant for the understanding of the interaction network in the active cavity. The structural variations induced by different inhibitors are examined. The effects of these interactions on the catalytic mechanism and on the binding of substrate are discussed. © 1992 Wiley‐Liss, Inc.
The refined crystal structure of Pseudomonas putida lipoamide dehydrogenase complexed with NAD+ at 2.45 Å resolution
Authors: Mattevi Andrea , Obmolova Galya , Sokatch John R. , Betzel Christian , Hol Wim G. J.
Published: May 2005

The three‐dimensional structure of one of the three lipoamide dehydroge‐nases occurring in Pseudomonas putida, LipDH Val, has been determined at 2.45 Å resolution. The orthorhombic crystals, grown in the presence of 20 mM NAD+, contain 458 residues per asymmetric unit. A crystallographic 2‐fold axis generates the dimer which is observed in solution. The final crystallographic R‐factor is 21.8% for 18,216 unique reflections and a model consisting of 3,452 protein atoms, 189 solvent molecules and 44 NAD+ atoms, while the overall B‐factor is unusually high; 47 Å2.

The structure of LipDH Val reveals the conformation of the C‐terminal residues which fold “back” into the putative lipoamide binding region. The C‐terminus has been proven to be important for activity by site‐directed mutagene‐sis. However, the distance of the C‐terminus to the catalytically essential residues is surprisingly large, over 6 Å, and the precise role of the C‐terminus still needs to be elucidated.

In this crystal form LipDH Val contains one NAD+ molecule per subunit. Its adenine‐ribose moiety occupies an analogous position as in the structure of glutathione reductase. However, the nicotinamide‐ribose moiety is far removed from its expected position near the isoallox‐azine ring and points into solution.

Comparison of LipDH Val with Azotobacter vinelandii lipoamide dehydrogenase yields an rms difference of 1.6 Å for 440 well defined Cα atoms per subunit. Comparing LipDH Val with glutathione reductase shows large differences in the tertiary and quaternary structure of the two enzymes. For instance, the two subunits in the dimer are shifted by 6 Å with respect to each other. So, LipDH Val confirms the surprising differences in molecular architecture between glutathione reductase and lipoamide dehydrogenase, which were already observed in Azotobacter vinelandii LipDH. This is the more remarkable since the active sites are located at the subunit interface and are virtually identical in all three enzymes. © 1992 Wiley‐Liss, Inc.
A systematic search for protein signature sequences
Authors: Sheridan Robert P. , Venkataraghavan R.
Published: May 2005

Signature sequences are contiguous patterns of amino acis 10‐50 resiues long that are associated with a particular structure or function in proteins. These may be of three types (by our nomenclature): superfamily signatures, remnant homologies, and motifs. We have performed a systematic search through a database of protein sequences to automatically and preferentially find remnant homologies and motifs. This was accomplished in three steps:

We generated a nonredundant sequence database.

We used BLAST3 (Altschul and Lipman, Proc. Natl. Acad. Sci. U.S.A. 87:5509–‐5513, 1990) to generate local pairwise and triplet sequence alignments for every protein in the database vs. every other.

We selected “interesting” alignments and grouped them into clusters. We find that most of the clusters contain segments from proteins which share a common structure or function. Many of them correspond to signatures previously noted in the literature. We discuss three previously recognized motifs in detail (FAD/NAD‐binding, ATP/GTP‐binding, and cytochrome b5‐like domains) to demonstrate how the alignments generated by our procedure are consistent with previous work and make structural and functional sense. We also discuss two signatures (for N‐acetyltransferases and glycerol‐phosphate binding) which to our knowledge have not been previously recognized. © 1992 Wiley‐Liss, Inc.
Fast structure alignment for protein databank searching
Authors: Orengo Christine A. , Brown Nigel P. , Taylor William R.
Published: May 2005

A fast method is described for searching and analyzing the protein structure databank. It uses secondary structure followed by residue matching to compare protein structures and is developed from a previous structural alignment method based on dynamic programming.

Linear representations of secondary structures are derived and their features compared to identify equivalent elements in two proteins. The secondary structure alignment then constrains the residue alignment, which compares only residues within aligned secondary structures and with similar buried areas and torsional angles. The initial secondary structure alignment improves accuracy and provides a means of filtering out unrelated proteins before the slower residue alignment stage.

It is possible to search or sort the protein structure databank very quickly using just secondary structure comparisons. A search through 720 structures with a probe protein of 10 secondary structures required 1.7 CPU hours on a Sun 4/280. Alternatively, combined secondary structure and residue alignments, with a cutoff on the secondary structure score to remove pairs of unrelated proteins from further analysis, took 10.1 CPU hours. The method was applied in searches on different classes of proteins and to cluster a subset of the databank into structurally related groups. Relationships were consistent with known families of protein Structure. © 1992 Wiley‐Liss, Inc.
Functional roles of amino acid residues involved in forming the α–helix‐turn–α–helix operator DNA binding motif of tet represser from Tn10
Authors: Baumeister Ralf , Müller Gerhard , Hecht Brigitte , Hillen Wolfgang
Published: May 2005

The Tn10derived Tet represser contains an amino acid segment with high homology to the α‐helix–turn–α‐helix motif (HTH) of other DNA binding proteins. The five most conserved amino acids in HTH are probably involved in structural formation of the motif. Their functional role was probed by saturation mutagenesis yielding 95 single amino acid replacement mutants of Tet repressor. Their binding efficiencies to tet operator were quantitatively determined in vivo. All functional mutants contain amino acid substitutions consistent with their proposed role in a HTH. In particular, only the two smallest amino acids (serine, glycine) can substitute a conserved alanine in the proposed first α‐helix without loss of activity. The last position of the first α‐helix, the second position in the turn, and the fourth position in the second α‐helix require mostly hydrophobic residues. The proposed C‐terminus of the first α‐helix is supported by a more active asparagine compared to glutamine replacement mutant of the wt leucine residue. The turn is located close to the protein surface as indicated by functional lysine and arginine replacements for valine. A glycine residue at the first position in the turn can be replaced by any amino acid yielding mutants with at least residual tet operator affinity. A structural model of the HTH of Tet repressor is presented. © 1992 Wiley‐Liss, Inc.
Structural principles for the propeller assembly of β‐sheets: The preference for seven‐fold symmetry
Authors: Murzin Alexey G. , Murzin Alexey G.
Published: May 2005

Twisted β‐sheets, packed face to face, may be arranged in circular formation like blades of a propeller or turbine. This β‐pro‐peller fold has been found in three proteins: that in neuraminidase consists of six β‐sheets while those in methylamine dehydrogenase and galactose oxidase are composed of seven β‐sheets. A model for multisheet packing in the β‐propeller fold is proposed. This model gives both geometrical parameters of the β‐propellers composed of different numbers of sheets and patterns of residue packing at their sheet‐to‐sheet interfaces. All the known β‐propeller structures have been analyzed, and the observed geometries and residue packing are found to be in good agreement with those predicted by models. It is shown that unusual seven‐fold symmetry is preferable to six‐ or eight‐fold symmetry for propeller‐like multisheet assembly. According to the model, a six β‐sheet propeller has to have predominantly small residues in the β‐strands closed to its sixfold axis, but no strong sequence constraints are necessary for a seven‐fold β‐propeller. © 1992 Wiley‐Liss, Inc.
Fast and simple monte carlo algorithm for side chain optimization in proteins: Application to model building by homology
Authors: Holm Lisa , Sander Chris
Published: May 2005

An unknown protein structure can be predicted with fair accuracy once an evolutionary connection at the sequence level has been made to a protein of known 3‐D structure. In model building by homology, one typically starts with a backbone framework, rebuilds new loop regions, and replaces nonconserved side chains. Here, we use an extremely efficient Monte Carlo algorithm in rotamer space with simulated annealing and simple potential energy functions to optimize the packing of side chains on given backbone models. Optimized models are generated within minutes on a workstation, with reasonable accuracy (average of 81% side chain χ1 dihedral angles correct in the cores of proteins determined at better than 2.5 Å resolution). As expected, the quality of the models decreases with decreasing accuracy of backbone coordinates. If the backbone was taken from a homologous rather than the same protein, about 70% side chain X1 angles were modeled correctly in the core in a case of strong homology and about 60% in a case of medium homology. The algorithm can be used in automated, fast, and reproducible model building by homology. © 1992 Wiley‐Liss, Inc.
Three‐dimensional structure of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans determined by molecular replacement at 2.8 Å resolution
Authors: Chen Longyin , Mathews F. Scott , Davidson Victor L. , Huizinga Eric G. , Vellieux Frederic M. D. , Hol Wim G. J.
Published: May 2005

The three‐dimensional structure of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans (PD‐MADH) has been determined at 2.8 Å resolution by the molecular replacement method combined with map averaging procedures, using data collected from an area detector. The structure of methylamine dehydrogenase from Thio‐bacillus versutus, which contains an “X‐ray” sequence, was used as the starting search model. MADH consists of 2 heavy (H) and 2 light (L) subunits related by a molecular 2‐fold axis. The H subunit is folded into seven four‐stranded β‐segments, forming a disk‐shaped structure, arranged with pseudo‐7‐fold symmetry. A 31‐residue elongated tail exists at the N‐terminus of the H subunit in MADH from T. versutus but is partially digested in this crystal form of MADH from P. denitrificans, leaving the H subunit about 18 residues shorter. Each L subunit contains 127 residues arranged into 10 β‐strands connected by turns. The active site of the enzyme is located in the L subunit and is accessible via a hydrophobic channel between the H and L subunits. The redox cofactor of MADH, tryptophan tryptophylquinone is highly unusual. It is formed from two co‐valently linked tryptophan side chains at positions 57 and 107 of the L subunit, one of which contains an orthoquinone. © 1992 Wiley‐Liss, Inc.
Structure of a rhombohedral R6 insulin/phenol complex
Authors: Smith G. David , Dodson Guy G.
Published: May 2005

Hexameric insulin has been crystallized from different conditions ina variety of crystalline modifications. In the presence of ∼1%phenol and at a pH of 8.5, a new rhom‐bohedral form is produced, space group R3, a = 79.92 Åand c = 40.39 Å, in whichthe asymmetric unit consists of a dimer. The structur has been solved and refined, using data between 8.0 and 2.5 Å resolution, to a residual of0.157. The two monomers in the asymmetric unit have nearly identical R conformations, that is, residues Bl through B8 are α‐helical, producing a continuous α‐helix from Bl through B19. A phenol molecule is hydrogen bonded to the carbonyl oxygen of A6 Cys of each monomer. Small differences in conformation and the final (2Fo‐Fc) and difference electron density maps suggest that an additional phenol molecule is coordinated to one of the two zinc ions.
Pitch diversity in α‐helical coiled coils
Authors: Seo John , Cohen Carolyn
Published: May 2005

Two complementary methods for measuring local pitch based on heptad position in α‐helical coiled coils are described and applied to six crystal structures. The results reveal a diversity of pitch values: two‐stranded coiled coils appear to have pitch values near 150 Å the values for three‐ and four‐stranded coiled coils range closer to 200 Å. The methods also provide a rapid and sensitive gauge of local coiled‐coil conformation. Polar or charged residues in the apolar interface between coiled‐coil helices markedly affect local pitch values, suggesting a connection between pitch uniformity and coiled‐coil stability. Moreover, the identification of a skip residue (heptad frame shift) in the hemaglutinin glycoprotein of influenza virus (HA) allows interpretation of local pitch changes. These results on relatively short coiled‐coil structures have relevance for the much longer fibrous proteins (many of which have skip residues) whose detailed structures are not yet established. We also show that local pitch values from molecular dynamics predictions of the GCN4 leucine zipper are in striking agreement with the high‐resolution crystal structure—a result not readily discerned by direct comparison of atomic coordinates. Taken together, these methods reveal specific aspects of coiled‐coil structure which may escape detection by global analyses of pitch. © 1993 Wiley‐Liss, Inc.
βVI Turns in peptides and proteins: A model peptide mimicry
Authors: Müller Gerhard , Gurrath Marion , Kurz Michael , Kessler Horst
Published: May 2005

To investigate the role of proline in defining β turn conformations within cyclic hexa‐ and pentapeptides we synthesized and determined the conformations of a series of L‐ and D‐proline‐containing peptides by means of 2D NMR spectroscopy and restrained molecular dynamics simulations. Due to cis/trans isomerism the L‐proline peptides adopt at least two different conformations that are analyzed and compared to the structures of the corresponding D‐proline peptides. The cis conformations of the compounds cyclo(‐Pro‐Ala‐Ala‐Pro‐Ala‐Ala‐), cyclo(‐Arg‐Gly‐Asp‐Phe‐Pro‐Gly‐), cyclo(‐Arg‐Gly‐Asp‐Phe‐Pro‐Ala‐), cyclo(‐Pro‐Ala‐Ala‐Ala‐Ala‐‐), and cyclo(‐Pro‐Ala‐Pro‐Ala‐Ala‐) form uncommon βVI turns that mimic the turn geometries found in crystallographically refined protein structures at such a detailed level that even preferred side chain orientations are reproduced. The ratios of the cis/trans isomers are analyzed in terms of the steric demand of the proline‐following residue. The conformational details derived from this study illustrate the importance of the examination of small model compounds derived from protein loop regions, especially if bioactive recognition sequences, such as RGD (Arg‐Gly‐Asp), are incorporated. © 1993 Wiley‐Liss, Inc.
A novel computer modeling approach to the structures of small bioactive peptides: The structure of gonadotropin releasing hormone
Authors: Gupta Hema M. , Talwar Gursaran P. , Salunke Dinakar M.
Published: May 2005

A novel computer modeling approach suitable for the structure analysis of small bioactive peptides has been developed. This approach involves identification of conformational patterns in protein structure data bank based on the sequence homology with the bioactive peptide. The models built on the basis of this homology and having common conformational patterns are analyzed under the structural constraints derived from the activity data of various synthetic analogs of the peptide. Application of this procedure to the gonadotropin releasing hormone (GnRH) resulted in a library of possible structures for GnRH, 9 among which shared a common β‐turn. Further analysis of the structures containing the β‐turn motif, in the context of the structure–activity data, led to a model for the active conformation of GnRH. The topology of the putative receptor binding site of the hormone is defined by a contiguous surface formed through an appropriate juxtaposition of the N‐terminal pGlu1 the guanidyl group of Arg8, aromatic side chain of Trp3, and the Gly10‐NH2 at the C‐terminal end. ©Wiley‐Liss, Inc.
An empirical energy function for threading protein sequence through the folding motif
Authors: Bryant Stephen H. , Lawrence Charles E.
Published: May 2005

In this paper we present a new residue contact potantial derived by statistical analysis of protein crystal structures. This gives mean hydrophobic and pairwise contact energies as a function of residue type and distance interval. To test the accuracy of this potential we generate model structures by “threading” different sequences through backbone folding motifs found in the structural data base. We find that conformational energies calculated by summing contact potentials show perfect specificity in matching the correct sequences with each globular folding motif in a 161‐protcin data set. They also identify correct models with the core folding motifs of heme‐rythrin and immunoglobulin McPC603 V1‐do‐ main, among millions of alternatives possible when we align subsequences with α‐helices and β‐strands, and allow for variation in the lengths of intervening loops. We suggest that contact potentials reflect important constraints on nonbonded interaction in native proteins, and that “threading” may be useful for structure prediction by recognition of folding motif. © 1993 Wiley‐Liss, Inc.
Site specific point mutation changes specificity: A molecular modeling study by free energy simulations and enzyme kinetics of the thermodynamics in ribonuclease T1 substrate interactions
Authors: Elofsson Arne , Kulinski Tadeusz , Rigler Rudolf , Nilsson Lennart
Published: May 2005

We have theoretically and experimentally studied the binding of two different ligands to wild‐type ribonuclease T1 (RNT1) and to a mutant of RNT1 with Glu‐46 replaced by Gln. The binding of the natural substrate 3′‐GMP has been compared with the binding of a fluorescent probe, 2‐aminopurine 3′‐monophosphate (2AP), and relative free energies of binding of these ligands to the mutant and the wild‐type (wt) enzyme have been calculated by free energy perturbation methods. The free energy perturbations predict that the mutant RNT1‐Gln‐46 binds 2AP better than 3′GMP, in agreement with experiments on dinucleotides. Four free energy perturbations, forming a closed loop, have been performed to allow the detection of systematic errors in the simulation procedure. Because of the larger number of atoms involved, it was necessary to use a much longer simulation time for the change in the protein, i.e., the perturbation from Glu to Gln, than in the perturbation from 3′‐GMP to 2AP. Finally the structure of the binding site is analyzed for understanding differences in catalytic speed and binding strength. © 1993 Wiley‐Liss, Inc.
Binding of cyanide, cyanate, and thiocyanate to human carbonic anhydrase II
Authors: Peng Z. , Merz Kenneth M. , Banci Lucia
Published: May 2005

Computer simulation techniques are used to address the question of how cyanide and related ions interact with human carbonic anhydrase II (HCAII). Spectroscopic results have suggested that cyanide is coordinated with the zinc ion, while recent X‐ray results suggest that the cyanide ion is noncovalently associated with the zinc–water or zinchydroxide form of the enzyme. We have carried out simulations on three models in an attempt to shed light on why the spectroscopic and X‐ray results differ. The first model we studied (Model I) has cyanide directly coordinated to the zinc ion, the second has it noncovalently interacting with the zinc–hydroxide (high pH) form of the enzyme (Model II), and the third has cyanide noncovalently interacting with the zinc–water (low pH) form of the enzyme (Model III). None of these models is satisfactory in explaining the available structural data obtained from X‐ray crystallography. This leads us to propose an alternative model, in which HCAII hydrates HCN to form an OH−/HCN complex coordinated to the Zn ion. Ab initio calculations are consistent with this model. Based on these results we are able to explain the observed crystallographic behavior of cyanate and, by inference, thiocyanate. © 1993 Wiley‐Liss, Inc.
Crystal structure of the complex of human α‐thrombin and nonhydrolyzable bifunctional inhibitors, hirutonin—2 and hirutonin—6
Authors: Zdanov Alexander , Wu Shan , DiMaio John , Konishi Yasuo , Li Yunge , Wu Xinjie , Edwards Brian F. P. , Martin Philip D. , Cygler Miroslaw
Published: May 2005

The crystal structure of the complexes of hirutonin‐2 and hirutonin‐6 with human α‐thrombin have been solved and refined to R‐factors of 0.169 (2.0 Å resolution) and 0.162 (201Å), respectively. Hirutonins belong to a family of bifunctional inhibitors bearing a noncleavable moiety mimicking the scissile bond. Hirutonin‐2 is an analog of (D)Phe‐Pro‐Arg‐Gly‐hirudin49–65; hirutonin‐6 has the same N‐terminal tripeptide connected to a shortened fibrinogen exosite‐binding part by a short, non‐peptidyl linker. The hirutonin‐6 molecule is well defined in the electron density with the exception of the C‐terminal Leu‐h61. The linker follows near the bottom of the canyon connecting the active site with the exosite, forms a short antiparallel β‐sheet‐like arrangement with Leu‐40‐Leu41 and makes van der Waals contacts with Glu39‐Leu40‐Leu41 of thrombin. In the thrombin‐hirutonin‐2 complex, the N‐ and C‐terminal parts of the inhibitor are well or dered (except the C‐terminal Gln‐h65) while the central portion of the linker is partially disordered. The glycine analog in the P1′ position of hirutonin‐2 assumes a conformation similar to that of the canonical form (Bode and Huber (1992) Eur. J. Biochem. 204 : 433‐451) and supports the identification of the S1′ site as restricted by His57, Trp60D, Lys60F, and the Cys42‐Cys58 disulfide bridge. The carbonyl oxygen of the P1 arginine residue is located in the oxyanion hole formed by the NH groups of Gly193 and Ser195, while the carbonyl carbon is positioned within a short distance, 2.8 Å, from the Oγ of Ser195. This resembles the conformation of the substrate‐like inhibitors bound to other serine proteases. The N‐terminal (D)Phe‐pro‐Arg fragment common to both inhibitors binds to thrombin in a fashion very similar to that of other inhibitors having this motif. The binding of the C‐terminus of hirutonins to the fibrinogen‐binding exosite is similar to that observed in hirudin and hirulog complexes. © 1993 Wiley‐Liss, Inc.
Single‐site modifications of half‐ligated hemoglobin reveal autonomous dimer cooperativity within a quaternary T tetramer
Authors: LiCata Vince J. , Dalessio Paula M. , Ackers Gary K.
Published: May 2005

The patterns of energetic response elicited by single‐site hemoglobin mutations and chemical mocdifications have been determined in order to probe the dimer–dimer interface of the half‐ligated tetramer (species[21]) that was previously shown to behave as allosterically distinct from both the unligated and fully ligated molecules1. In this study the free energies of quaternary assembly(dimers to tetramers) were determined for aseries of 24 tetrameric species in which one dimeric half‐molecule is ligated while the adjacent αβ dimer is unligated and contains a single amino acid modification. Assembly energies have also been determined for tetramers bearing the same amino acid modifications but where the hemesites were completely vacant and additionally where they were fully occupied. A total of 72 molecular species were thus characterized. It was found that mutationally induced perturbations to the free energy of quaternary assembly were identical for the half‐ligated tetramers and the unligated tetramers over the entire spatial distrubution of altered sites, but exhibited a radically different pattern from that of the fully ligated molecules. These results indicate that the dimer–dimer interface of the half‐ligated tetramer(species[21]) has the same quaternary sturcture as that of the unligated molecule, i.e, “quaternary T.” This quaternary structure assignment of species [21] strongly supports the operation of a Symmetry Rule which translates changes in hemesite ligation into six T → R quaternary switchpoints2. Analysis of the observed Symmetry Rule behaviour in relation to the measured distribution of cooperative free energies for the partially ligated species reveals significant cooperativity between α and β subunits of the dimeric half‐tetramer within quaternary T. The mutational results indicate that these interactions are not “paid for” by breaking or making noncovalent bonds at the dimer–dimer interface (α1β2). They arise from structural and energetic changes that are “internal” to the ligated dimer even though its association with the unligated dimer is required for the cooperativity to occur. Free energy of “tertiary constraint” is thus generated by the first binding step and is propagated to the second hemesite while the dimer–dimer interface α1β2serves as a constraint. The “sequential” cooperativity that occurs within the half‐molecule is thus preconditioned by the constraint of a quaternary T interface; release of this constraint by dissociation produces only noncooperative dimers. When the constraint is released functionally by T to R dimer rearrangement (at each switch‐point specified by the a Symmetry Rule) the alterations of interfacial bonds then dominate the energetics of cooperativity. © 1993 Wiley‐Liss, Inc.
A calculation strategy for the structure determination of symmetric demers by 1H NMR
Authors: Nilges Michael
Published: May 2005

The structure determination of symmetric dimers by NMR is impeded by the ambiguity of inter‐ and intramonomer NOE crosspeaks. In this paper, a calculation strategy is presented that allows the calculation of dimer structures without resolving ther ambuguity by additional experiments (like asymmetric labeling). The strategy employs a molecular dynamic‐based simulated annealing approach to minimize a traget function. The experimental part of the target function contains distance restraints that correctly describe the ambiguity of the NOE peaks, and a novel term that restrains the symmetry of the dimer without requiring the knowledge of the symmetry axis. The use of the method is illustrated by three examples, using experimentally obtained data and model data derived from a known structure. For the purpose of testing the method, it is assumed that every NOE crosspeak is ambiguous in all three cases. It is shown that the structure of a homologous protein is known and in ab intio structure determination. The method can be extended to higher order symmetric multimers. © 1993 Wiley‐Liss, Inc.
Preparation and characterization of the E168Q site‐directed mutant of yeast enolase 1
Authors: Brewer John M. , Robson Robert L. , Glover Claiborne V. C. , Holland Michael J. , Lebioda Lukasz
Published: May 2005

Yeast has two enolase isozymes (called 1 and 2), either of which suffices for growth. We cloned DNA encoding the enolase 1 protein coding and promoter regions flanked by BamHI termini using the PCR. The DNA, which contained no nucleotide base changes altering the protein sequence, was cloned into the multicopy shuttle vector pRS314 and transformed into a yeast strain with a deletion in its enolase 1 gene. The resulting plasmid‐containing strain makes enolase 1 in quantities which depend on cell growth. A ‘charge shuttle’ mechanism of action of enolase based on X‐ray crystallographic evidence (Lebioda and Stec, Biochemistry 30 : 2817, 1991) involves Glu‐168 accepting a proton from a water molecule that in turn accepts a proton from a carbon‐2 of the substrate. We prepared the E168Q mutant of enolase 1 by oligonucleotide‐directed site‐directed mutagenesis. Its identity was confirmed by N‐terminal sequence analysis, HPLC on Superose 12, SDS‐gel electrophoresis, and the sequence of the mutated DNA protein‐coding region. The E168Q mutant has approximately 0.01% of the activity of native enolase. It binds substrate/product, AEP (3‐aminoenolpyruvate‐2‐phosphate, the 3‐amino analogue of the product phosphoenolpyruvate) and TSP (D‐tartronate semialdehyde‐2‐phosphate, the aldehyde analogue of the substrate 2‐phosphoglycerate), the latter two at least with affinities similar to those of the native enzyme. The E168Q enolase also produces absorbance changes in the analogues. The reaction with AEP is consistent with the ‘charge shuttle’ mechanism; the reaction with TSP, which presumably requires proton removal from carbon‐2, is complex but shows a very slow phase consistent with expectations. © 1993 Wiley‐Liss, Inc.
Entropy in biological binding processes: Estimation of translational entropy loss
Authors: Murphy Kenneth P. , Xie Dong , Thompson Kelly S. , Amzel L. Mario , Freire Ernesto
Published: May 2005

The loss of translational degrees of freedom makes an important, unfavorable contribution to the free energy of binding. Examination of experimental values suggest that calculation of this entropy using the Sackur–Tetrode equation produces largely overestimated values. Better agreement is obtained using the cratic entropy. Theoretical considerations suggest that the volumes available for the movement of a ligand in solution and in a complex are rather similar, suggesting also that the cratic entropy provides the best estimate of the loss of translational entropy. © 1994 John Wiley & Sons, Inc.
A Connected‐cluster of hydration around myoglobin: Correlation between molecular dynamics simulations and experiment
Authors: Lounnas Valère , Pettitt B. Montgomery
Published: May 2005

An analysis of a molecular dynamics simulation of metmyoglobin in an explicit solvent environment of 3,128 water molecules has been performed. Both statics and dynamics of the protein‐solvent interface are addressed in a comparison with experiment. Three‐dimensional density distributions, temperature factors, and occupancy weights are computed for the solvent by using the trajectory coordinates. Analysis of the hydration leads to the localization of more than 500 hydration sites distributed into multiple layers of solvation located between 2.6 and 6.8 Å from the atomic protein surface. After locating the local solvent density maxima or hydration sites we conclude that water molecules of hydration positions and hydration sites are distinct concepts. Both global and detailed properties of the hydration cluster around myoglobin are compared with recent neutron and X‐ray data on myoglobin. Questions arising from differences between X‐ray and neutron data concerning the locations of the protein‐bound water are investigated. Analysis of water site differences found from X‐ray and neutron experiments compared with our simulation shows that the simulation gives a way to unify the hydration picture given by the two experiments. © 1994 John Wiley & Sons, Inc.
Distribution function implied dynamics versus residence times and correlations: Solvation shells of myoglobin
Authors: Lounnas Valère , Pettitt B. Montgomery
Published: May 2005

The dynamics of water at the protein–solvent interface is investigated through the analysis of a molecular dynamics simulation of metmyoglobin in explicit aqueous environment. Distribution implied dynamics, harmonic and quasiharmonic, are compared with the simulated macroscopic dynamics. The distinction between distinguishable solvent molecules and hydration sites developed in the previous paper is used. The simulated hydration region within 7 Å from the protein surface is analyzed using a set of 551 hydration sites characterized by occupancy weights and temperature B‐factors determined from the simulation trajectory. The precision of the isotropic harmonic and anisotropic harmonic models for the description of proximal solvent fluctuations is examined. Residence times and dipole reorientation times of water around the protein surface are compared with NMR and ESR results. A correlation between diffraction experiment quantities such as the occupancy weights and temperature factors and the residence and correlation times resulting from magnetic resonance experiments is found via comparison with simulation. © 1994 John Wiley & Sons, Inc.
Crystallization and preliminary x‐ray diffraction studies of a monoclonal antibody fab fragment against foot‐and‐mouth disease virus and of its complex with the main antigenic site peptide
Authors: Verdaguer Nuria , Mateu Mauricio G. , Bravo Jerónimo , Tormo José , Giralt Ernest , Andreu David , Domingo Esteban , Fita Ignacio
Published: May 2005

The Fab fragment of the neutralizing monoclonal antibody SD6 elicited against foot‐and‐mouth disease virus (FMDV) C‐SBcl and its complex with a peptide, corresponding to the major antigenic site of FMDV (VPl residues 136–150, YTASARGDLAHLTTT), have been crystallized using the hanging drop vapor diffusion techniques. For the isolated Fab, crystals diffracting to 2.5 Å resolution were obtained at room temperature using ammonium sulfate as precipitant. These crystals are monoclinic, space group C2, and unit cell parameters a = 109.53 Å, b = 89.12 Å, c = 64.04 Å, and β = 112.9° and contain one Fab molecule per asymmetric unit. Crystals from the complex diffract, at least, to 2.8 Å resolution and were obtained, at room temperature, using PEG as precipitant. These crystals are monoclinic, space group P2, and unit cell parameters a = 56.11 Å, b = 60.67 Å, c = 143.45 Å, and β = 95.4°, Density packing considerations indicate that there are two Fab molecules in the asymmetric unit. © 1994 John Wiley & Sons, Inc.
Treatment of electrostatic effects in proteins: Multigrid‐based newton iterative method for solution of the full nonlinear poisson–boltzmann equation
Authors: Holst Michael , Kozack Richard E. , Saied Faisal , Subramaniam Shankar
Published: May 2005

The nonlinear Poisson–Boltzmann equation (NPBE) provides a continuum description of the electrostatic field in an ionic medium around a macromolecule. Here, a novel approach to the solution of the full NPBE is developed. This robust and efficient algorithm combines multilevel techniques with a damped inexact Newton's method. The CPU time required for solution of the full NPBE, which is less than that for standard single‐grid approaches in solving the corresponding linearized equation, is proportional to the number of unknowns enabling applications to very large macromolecular systems. Convergence of the method is demonstrated for a variety of protein systems. Comparison of the solutions to the linearized Poisson–Boltzmann equation shows that the damping of the electrostatic field around the charge is increased and that the potential scales logarithmically with charge. The inclusion of the full nonlinearity thus reduces the impact of highly charged residues on protein surfaces and provides a more realistic representation of electrostatic effects. This is demonstrated through calculation of potential around the active site regions of the 1,266‐residue tryptophan synthase dimer and in the computation of rate constants from Brownian dynamics calculations in the superoxide dismutase–superoxide and antibody–antigen systems. © 1994 John Wiley & Sons, Inc.
Cyanomet human hemoglobin crystallized under physiological conditions exhibits the Y quaternary structure
Authors: Smith Francine R. , Simmons Katharine C.
Published: May 2005

Cyanomet human hemoglobin has been crystallized at a chloride ion concentration and pH similar to physiological conditions. Molecular replacement calculations definitively show that the hemoglobin subunits are arranged in the Y quaternary form recently discovered in carbon monoxy hemoglobin Ypsilanti (99 Asp–Tyr), and subsequently observed in carbon monoxy normal human hemoglobin crystallized at low ionic strength and low pH. The structure has been refined at 2.09 Å resolution to an R‐value of 0.232, and further refinement is currently underway. Although the refinement is not yet complete, our results are the first indication that the Y structure may represent an important quaternary form of liganded hemoglobin under physiological buffer conditions. These results suggest the need for a reexamination of structure–function correlations in the hemoglobin system. © 1994 John Wiley & Sons, Inc.
α‐Helix‐forming propensities in peptides and proteins
Authors: Creamer Trevor P. , Rose George D.
Published: May 2005

Much effort has been invested in seeking to understand the thermodynamic basis of helix stability in both peptides and proteins. Recently, several groups have measured the helix‐forming propensities of individual residues (Lyu, P. C., Liff, M. I., Marky, L. A., Kallenbach, N. R. Science 250:669–673, 1990; O'Neil, K. T., DeGrado, W. F. Science 250:646–651, 1990; Padmanabhan, S., Marqusee, S., Ridgeway, T., Laue, T. M., Baldwin, R. L. Nature (London) 344:268–270, 1990). Using Monte Carlo computer simulations, we tested the hypothesis that these differences in measured helix‐forming propensity are due primarily to loss of side chain conformational entropy upon helix formation (Creamer, T. P., Rose, G. D. Proc. Natl. Acad. Sci. U.S.A. 89:5937–5941, 1992). Our previous study employed a rigid helix backbone, which is here generalized to a completely flexible helix model in order to ensure that earlier results were not a methodological artifact. Using this flexible model, side chain rotamer distributions and entropy losses are calculated and shown to agree with those obtained earlier. We note that the side chain conformational entropy calculated for Trp in our previous study was in error; a corrected value is presented. Extending earlier work, calculated entropy losses are found to correlate strongly with recent helix propensity scales derived from substitutions made within protein helices (Horovitz, A., Matthews, J. M., Fersht, A. R. J. Mol. Biol. 227:560–568, 1992; Blaber, M., Zhang, X.‐J., Matthews, B. M. Science 260:1637–1640, 1993). In contrast, little correlation is found between these helix propensity scales and the accessible surface area buried upon formation of a model polyalanyl α‐helix. Taken in sum, our results indicate that loss of side chain entropy is a major determinant of the helix‐forming tendency of residues in both peptide and protein helices. © 1994 Wiley‐Liss, Inc.
Crystallization and characterization of colicin E1 channel‐forming polypeptides
Authors: Elkins Patricia A. , Song Ho Yeong , Cramer William A. , Stauffacher Cynthia V.
Published: May 2005

Crystals of the channel‐forming domain of colicin E1 from E. coli were grown by vapor diffusion at pH 6.4 and higher pH values. Cleavage of the colicin molecule with trypsin or thermolysin produced two of the pore‐forming polypeptides used in these experiments. The third polypeptide was purified from a constructed plasmid that overexpresses only the C‐terminal domain of colicin E1. Polypeptide crystals are tetragonal with space group I4, have one monomer in the asymmetric unit, and diffract to 2.2–2.4 Å. Unit cell parameters for the tryptic and thermolytic polypeptides are a = 102.9 Å and c = 35.6 Å. Crystals of the overexpressed polypeptide have unit cell parameters of a =87.2 Å and c =59.1 Å. The crystals were characterized by precession photography, and native data sets of each channel‐forming fragment were collected on a Siemens‐Nicolet area detector. The crystallization and characterization of these polypeptides are the first steps in the structure determination of the channel‐forming domain of colicin E1. © 1994 Wiley‐Liss, Inc.
The closed conformation of a highly flexible protein: The structure of E. coli adenylate kinase with bound AMP and AMPPNP
Authors: Berry Michael B. , Meador Bill , Bilderback Tim , Liang Peng , Glaser Michael , Phillips George N.
Published: May 2005

The structure of E. coli adenylate kinase with bound AMP and AMPPNP at 2.0 Å resolution is presented. The protein crystallizes in space group C2 with two molecules in the asymmetric unit, and has been refined to an R factor of 20.1% and an Rfree of 31.6%. In the present structure, the protein is in the closed (globular) form with the large flexible lid domain covering the AMPPNP molecule. Within the protein, AMP and AMPPNP, an ATP analog, occupy the AMP and ATP sites respectively, which had been suggested by the most recent crystal structure of E. coli adenylate kinase with AP5A bound (Müller and Schulz, 1992, ref. 1) and prior fluorescence studies (Liang et al., 1991, ref. 2). The binding of substrates and the positions of the active site residues are compared between the present structure and the E. coli adenylate kinase/Ap5A structure. We failed to detect a peak in the density map corresponding to the Mg2+ ion which is required for catalysis, and its absence has been attributed to the use of ammonium sulfate in the crystallization solution. Finally, a comparison is made between the present structure and the structure of the heavy chain of muscle myosin. © 1994 Wiley‐Liss, Inc.