bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural energetics of barstar studied by differential scanning microcalorimetry
Authors: Wintrode Patrick L. , Griko Yuri V. , Privalov Peter L. 
Published: February 2009

AbstractThe energetics of barstar denaturation have been studied by CD and scanning microcalorimetry in an extended range of pH and salt concentration. It was shown that, upon increasing temperature, barstar undergoes a transition to the denatured state that is well approximated by a two‐state transition in solutions of high ionic strength. This transition is accompanied by significant heat absorption and an increase in heat capacity. The denaturational heat capacity increment at ≈︁75 °C was found to be 5.6 ± 0.3 kJ K−1 mol−1. In all cases, the value of the measured enthalpy of denaturation was notably lower than those observed for other small globular proteins. In order to explain this observation, the relative contributions of hydration and the disruption of internal interactions to the total enthalpy and entropy of unfolding were calculated. The enthalpy and entropy of hydration were found to be in good agreement with those calculated for other proteins, but the enthalpy and entropy of breaking internal interactions were found to be among the lowest for all globular proteins that have been studied. Additionally, the partial specific heat capacity of barstar in the native state was found to be 0.37 ± 0.03 cal K−1 g−1, which is higher than what is observed for most globular proteins and suggests significant flexibility in the native state. It is known from structural data that barstar undergoes a conformational change upon binding to its natural substrate barnase. Our data, which indicate that barstar has a loosely packed interior, suggest that high conformational flexibility of barstar's native structure may play an important role in allowing it to optimize its contacts with barnase upon binding without disrupting favorable, tightly packed internal interactions.
Urea‐induced dissociation and unfolding of dodecameric glutamine synthetase from escherichia coli: Calorimetric and spectral studies
Authors: Zolkiewski Michal , Nosworthy Neil J. , Ginsburg Ann 
Published: February 2009

AbstractUrea‐induced dissociation and unfolding of manganese · glutamine synthetase (Mn‐GS) have been studied at 37 °C (pH 7) by spectroscopic and calorimetric methods. In 0 to ∼2 M urea, Mn‐GS retains its dodecameric structure and full catalytic activity. Mn‐GS is dissociated into subunits in 6 M urea, as evidenced by a 12‐fold decrease in 90° light scattering and a monomer molecular weight of 51,800 in sedimentation equilibrium studies. The light scattering decrease in 4 M urea parallels the time course of Trp exposure but occurs more rapidly than changes in secondary structure and Tyr exposure. Early and late kinetic steps appear to involve predominantly disruption of intra‐ring and inter‐ring subunit contacts, respectively, in the layered hexagonal structure of Mn‐GS. The enthalpies for transferring Mn‐GS into urea solutions have been measured by titration calorimetry. After correcting for the enthalpy of binding urea to the protein, the enthalpy of dissociation and unfolding of Mn · GS is 14 ± 4 cal/g. A net proton uptake of ∼50 H+/dodecamer accompanies unfolding reactions. The calorimetric data are consistent with urea binding to multiple, independent sites in Mn‐GS and the number of binding sites increasing ∼9‐fold during the protein unfolding.
Finding flexible patterns in unaligned protein sequences
Authors: Jonassen Inge , Collins John F. , Higgins Desmond G. 
Published: February 2009

AbstractWe present a new method for the identification of conserved patterns in a set of unaligned related protein sequences. It is able to discover patterns of a quite general form, allowing for both ambiguous positions and for variable length wildcard regions. It allows the user to define a class of patterns (e.g., the degree of ambiguity allowed and the length and number of gaps), and the method is then guaranteed to find the conserved patterns in this class scoring highest according to a significance measure defined. Identified patterns may be refined using one of two new algorithms. We present a new (nonstatistical) significance measure for flexible patterns. The method is shown to recover known motifs for PROSITE families and is also applied to some recently described families from the literature.
Multidomain organization of eukaryotic guanine nucleotide exchange translation initiation factor eIF‐2B subunits revealed by analysis of conserved sequence motifs
Authors: Koonin Eugene V. 
Published: February 2009

AbstractComputer‐assisted analysis of amino acid sequences using methods for database screening with individual sequences and with multiple alignment blocks reveals a complex multidomain organization of yeast proteins GCD6 and GCD1, and mammalian homolog of GCD6 — subunits of the eukaryotic translation initiation factor eIF‐2B involved in GDP/GTP exchange on eIF‐2. It is shown that these proteins contain a putative nucleotide‐binding domain related to a variety of nucleotidyltransferases, most of which are involved in nucleoside diphosphate‐sugar formation in bacteria. Three conserved motifs, one of which appears to be a variant of the phosphate‐binding site (P‐loop) and another that may be considered a specific version of the Mg2+‐binding site of NTP‐utilizing enzymes, were identified in the nucleotidyltransferase‐related domain. Together with the third unique motif adjacent to the P‐loop, these motifs comprise the signature of a new superfamily of nucleotide‐binding domains. A domain consisting of hexapeptide amino acid repeats with a periodic distribution of bulky hydrophobic residues (isoleucine patch), which previously have been identified in bacterial acetyltransferases, is located toward the C‐terminus from the nucleotidyltransferase‐related domain. Finally, at the very C‐termini of GCD6, eIF‐2Be, and two other eukaryotic translation initiation factors, eIF‐4γ and eIF‐5, there is a previously undetected, conserved domain. It is hypothesized that the nucleotidyltransferase‐related domain is directly involved in the GDP/GTP exchange, whereas the C‐terminal conserved domain may be involved in the interaction of eIF‐2B, eIF‐4γ, and eIF‐5 with eIF‐2.
Molecular model of the N‐terminal receptor‐binding domain of the human CD6 ligand ALCAM
Authors: Bajorath Jürgen , Bowen Michael A. , Aruffo Alejandro 
Published: February 2009

AbstractCD6‐ligand interactions have been implicated in the regulation of T‐cell adhesion and activation. CD6 is a member of the scavenger receptor family, whereas its human ligand (ALCAM) belongs to the immunoglobulin superfamily. The extracellular region of ALCAM includes five immunoglobulin‐like domains. As a fusion protein, the N‐terminal extracellular domain of ALCAM (ALCAMD1) binds specifically to CD6. We report the construction, assessment, and analysis of a molecular model of ALCAMD1. The model defines the CDR‐analogous loops, the location of N‐linked glycosylation sites, and residues that form the β‐sheet faces of the immunoglobulin‐like domain. Predicted structural characteristics of the A'GFCC “C” face of the model are consistent with the presence of monomeric and dimeric forms of ALCAMD1, which has implications for the receptor‐ligand interactions.
Crystallization of the chaperone protein SecB
Authors: Vrielink Alice , Beamer Lesa , Le Thanh , Eisenberg David 
Published: February 2009

AbstractThe secretory protein SecB found inEscherichia coliis a molecular chaperone that binds to precursor forms of a number of proteins targeted for export to the periplasmic space. SecB maintains these proteins in a translocation‐competent conformation facilitating the translocation process. The material has been cloned and expressed inE. coli. Crystals have been grown from polyethylene glycol 8000 by vapor diffusion using the hanging drop technique. These crystals are monoclinic, belonging to space group C2 with unit cell dimensionsa= 56.0 Å,b= 111.1 Å,c= 134.7 Å, and β = 104°. The crystals diffract to 8 Å resolution on a Rigaku imaging plate detector. Dynamic light scattering experiments suggest that SecB exhibits aggregation behavior with a number of different precipitating agents. These results may explain resistance of SecB to forming ordered crystals.
Structural analysis of zinc substitutions in the active site of thermolysin
Authors: Holland Debra R. , Hausrath Andrew C. , Juers Doug , Matthews Brian W. 
Published: February 2009

AbstractNative thermolysin binds a single catalytically essential zinc ion that is tetrahedrally coordinated by three protein ligands and a water molecule. During catalysis the zinc ligation is thought to change from fourfold to fivefold. Substitution of the active‐site zinc with Cd2+, Mn2+, Fe2+, and Co2+ alters the catalytic activity (Holmquist B, Vallee BL, 1974, J Biol Chem 249:4601–4607). Excess zinc inhibits the enzyme. To investigate the structural basis of these changes in activity, we have determined the structures of a series of metal‐substituted thermolysins at 1.7–1.9 Å resolution.The structure of the Co2+‐substituted enzyme is shown to be very similar to that of wild type except that two solvent molecules are liganded to the metal at positions that are thought to be occupied by the two oxygens of the hydrated scissile peptide in the transition state. Thus, the enhanced activity toward some substrates of the cobalt‐relative to the zinc‐substituted enzyme may be due to enhanced stabilization of the transition state. The ability of Zn2+ and Co2+ to accept tetrahedral coordination in the Michaelis complex, as well as fivefold coordination in the transition state, may also contribute to their effectiveness in catalysis. The Cd2+‐ and Mn2+‐substituted thermolysins display conformational changes that disrupt the active site to varying degrees and could explain the associated reduction of activity. The conformational changes involve not only the essential catalytic residue, Glu 143, but also concerted side‐chain rotations in the adjacent residues Met 120 and Leu 144. Some of these side‐chain movements are similar to adjustments that have been observed previously in association with the “hinge‐bending” motion that is presumed to occur during catalysis by the zinc endoproteases.In the presence of excess zinc, a second zinc ion is observed to bind at His 231 within 3.2 Å of the zinc bound to native thermolysin, explaining the inhibitory effect.
Stromelysin‐1: Three‐dimensional structure of the inhibited catalytic domain and of the C‐truncated proenzyme
Authors: Becker Joseph W. , Marcy Alice I. , Rokosz Laura L. , Axel Melinda G. , Burbaum Jonathan J. , Fitzgerald Paula M.D. , Cameron Patricia M. , Esser Craig K. , Hagmann William K. , Hermes Jeffrey D. , Springer James P. 
Published: February 2009

AbstractThe proteolytic enzyme stromelysin‐1 is a member of the family of matrix metalloproteinases and is believed to play a role in pathological conditions such as arthritis and tumor invasion. Stromelysin‐1 is synthesized as a proenzyme that is activated by removal of an N‐terminal prodomain. The active enzyme contains a catalytic domain and a C‐terminal hemopexin domain believed to participate in macromolecular substrate recognition. We have determined the three‐dimensional structures of both a C‐truncated form of the proenzyme and an inhibited complex of the catalytic domain by X‐ray diffraction analysis. The catalytic core is very similar in the two forms and is similar to the homologous domain in fibroblast and neutrophil collagenases, as well as to the stromelysin structure determined by NMR. The prodomain is a separate folding unit containing three α‐helices and an extended peptide that lies in the active site of the enzyme. Surprisingly, the amino‐to‐carboxyl direction of this peptide chain is opposite to that adopted by the inhibitor and by previously reported inhibitors of collagenase. Comparison of the active site of stromelysin with that of thermolysin reveals that most of the residues proposed to play significant roles in the enzymatic mechanism of thermolysin have equivalents in stromelysin, but that three residues implicated in the catalytic mechanism of thermolysin are not represented in stromelysin.
Water molecules participate in proteinase‐inhibitor interactions: Crystal structures of Leu18, Ala18, and Gly18 variants of turkey ovomucoid inhibitor third domain complexed with Streptomyces griseus proteinase B
Authors: Huang Kui , James Michael N.G. , Lu Wuyuan , Laskowski Michael , Anderson Stephen 
Published: February 2009

AbstractCrystal structures of the complexes of Streptomyces griseus proteinase B (SGPB) with three P1 variants of turkey ovomucoid inhibitor third domain (OMTKY3), Leu18, Ala18, and Gly18, have been determined and refined to high resolution. Comparisons among these structures and of each with native, uncomplexed SGPB reveal that each complex features a unique solvent structure in the S1 binding pocket. The number and relative positions of water molecules bound in the S1 binding pocket vary according to the size of the side chain of the P1 residue. Water molecules in the S1 binding pocket of SGPB are redistributed in response to the complex formation, probably to optimize hydrogen bonds between the enzyme and the inhibitor. There are extensive water‐mediated hydrogen bonds in the interfaces of the complexes. In all complexes, Asn 36 of OMTKY3 participates in forming hydrogen bonds, via water molecules, with residues lining the S1 binding pocket of SGPB. For a homologous series of aliphatic straight side chains, Gly18, Ala18, Abu18, Ape18, Ahx18, and Ahp18 variants, the binding free energy is a linear function of the hydrophobic surface area buried in the interface of the corresponding complexes. The resulting constant of proportionality is 34.1 cal mol−1 Å−2. These structures confirm that the binding of OMTKY3 to the preformed S1 pocket in SGPB involves no substantial structural disturbances that commonly occur in the site‐directed mutagenesis studies of interior residues in other proteins, thus providing one of the most reliable assessments of the contribution of the hydrophobic effect to protein‐complex stability.
Thermal stability determinants of chicken egg‐white lysozyme core mutants: Hydrophobicity, packing volume, and conserved buried water molecules
Authors: Shih Phoebe , Kirsch Jack F. , Holland Debra R. 
Published: February 2009

AbstractA series of 24 mutants was made in the buried core of chicken lysozyme at positions 40, 55, and 91. The midpoint temperature of thermal denaturation transition (Tm) values of these core constructs range from 60.9 to 77.3 °C, extending an earlier, more limited investigation on thermostability. The Tm values of variants containing conservative replacements for the wild type (WT) (Thr 40‐Ile 55‐Ser 91) triplet are linearly correlated with hydrophobicity (r = 0.81) and, to a lesser degree, with combined side‐chain volume (r = 0.75). The X‐ray structures of the S91A (1.9 Å) and I55L/S91T/D101S (1.7 Å) mutants are presented. The former amino acid change is found in duck and mammalian lysozymes, and the latter contains the most thermostable core triplet. A network of four conserved, buried water molecules is associated with the core. It is postulated that these water molecules significantly influence the mutational tolerance at the individual triplet positions. The pH dependence of Tm for the S91D mutant was compared with that of WT enzyme. The pKa of S91D is 1.2 units higher in the native than in the denatured state, corresponding to ΔΔG298 = 1.7 kcal/mol. This is a low value for charge burial and likely reflects the moderating influence of the buried water molecules or a conformational change. Thermal and chemical denaturation and far UV CD spectroscopy were used to characterize the in vitro properties of I55T. This variant, which buries a hydroxyl group, has similar properties to those of the human amyloidogenic variant I56T.
Atomic solvation parameters in the analysis of protein‐protein docking results
Authors: Cummings Maxwell D. , Read Randy J. , Hart Trevor N. 
Published: February 2009

AbstractSeveral sets of amino acid surface areas and transfer free energies were used to derive a total of nine sets of atomic solvation parameters (ASPs). We tested the accuracy of each of these sets of parameters in predicting the experimentally determined transfer free energies of the amino acid derivatives from which the parameters were derived. In all cases, the calculated and experimental values correlated well. We then chose three parameter sets and examined the effect of adding an energetic correction for desolvation based on these three parameter sets to the simple potential function used in our multiple start Monte Carlo docking method. A variety of protein‐protein interactions and docking results were examined. In the docking simulations studied, the desolvation correction was only applied during the final energy calculation of each simulation. For most of the docking results we analyzed, the use of an octanol‐water‐based ASP set marginally improved the energetic ranking of the low‐energy dockings, whereas the other ASP sets we tested disturbed the ranking of the low‐energy dockings in many of the same systems. We also examined the correlation between the experimental free energies of association and our calculated interaction energies for a series of proteinase‐inhibitor complexes. Again, the octanol‐water‐based ASP set was compatible with our standard potential function, whereas ASP sets derived from other solvent systems were not.
Thrombin‐binding affinities of different disulfide‐bonded isomers of the fifth EGF‐like domain of thrombomodulin
Authors: Hunter Michael J. , Komives Elizabeth A. 
Published: February 2009

AbstractThe fifth EGF‐like domain of thrombomodulin (TM), both with and without the amino acids that connect the fifth domain to the sixth domain, has been synthesized and refolded to form several different disulfide‐bonded isomers. The domain without the connecting region formed three disulfide‐bonded isomers upon refolding under redox conditions. Of these three isomers, the (1–2,3–4,5–6) bonded isomer was the best inhibitor of fibrinogen clotting and also of the thrombin‐TM interaction that results in protein C activation, but all the isomers were inhibitors in both assays. The isomer containing an EGF‐like disulfide‐bonding pattern (1–3,2–4,5–6) was not found among the oxidation products.The domain with the connecting region amino acids (DIDE) at the C‐terminus formed two isolable products upon refolding in redox buffer. These products had the same two disulfide‐bonding patterns as the earliest and latest eluting isomers of the domain without the DIDE. In order to compare the thrombin‐binding affinities of these isomers to the isomer with the EGF‐like disulfide bonds, acetamidomethyl protection of the second and fourth cysteines was used to force the disulfide bonds into the EGF‐like pattern. Thrombin‐binding affinity, measured as inhibition of fibrinogen clotting and as inhibition of protein C activation correlated inversely with the number of crossed disulfide bonds. As was found for the domain without the connecting region, the isomer that was the best inhibitor of fibrinogen clotting and of protein C activation was the isomer with no crossing disulfide bonds (1–2,3–4,5–6). This isomer doubled the clotting time at a concentration of 200 nM and showed a Ki for protein C activation of 2 μM, both an order of magnitude better than the isomer with EGF‐like disulfide bonds.
Production, purification, and crystallization of human interleukin‐1β converting enzyme derived from an Escherichia coli expression system
Authors: Malinowski John J. , Grasberger Bruce L. , Trakshel Gary , Huston Edward E. , Banks Tracey M. , Brake Patricia G. , Ciccarelli Richard B. , Jones Barry N. , Koehn James A. , Kratz Diane , Lundberg Nicole , Stevis Panayiotis E. , Helaszek Carla T. , Ator Mark A. , Small Wood Angela M. , Stams Travis , Rubin Byron , Alexander Richard S. 
Published: February 2009

AbstractInterleukin‐1β converting enzyme (ICE) is a cysteine protease that catalyzes the conversion of the inactive precursor form of IL‐1β to an active mature form. The mature form of IL‐1β is involved in mediating inflammatory responses and in the progression of autoimmune diseases. We recently reported on the production of active human ICE in insect cells using the baculovirus expression system (Wang XM et al., 1994, Gene 145:273–277). Because the levels of expression achieved with this system were limiting for the purpose of performing detailed biochemical and biophysical studies, we examined the production of ICE in Escherichia coli. By using a tac promoter‐based expression system and fusion to thioredoxin we were able to recover high levels of active ICE protein. The expressed protein, which was distributed between the soluble and insoluble fractions, was purified to homogeneity from both fractions using a combination of classical and affinity chromatography. Comparisons of ICE derived from both fractions indicated that they were comparable in their specific activities, subunit composition, and sensitivities to specific ICE inhibitors. The combined yields of ICE obtained from the soluble and insoluble fractions was close to 1 mg/L of induced culture. Recombinant human ICE was crystallized in the presence of a specific ICE inhibitor in a form suitable for X‐ray crystallographic analysis. This readily available source of ICE will facilitate the further characterization of this novel and important protease.
Homology modeling of an immunoglobulin‐like domain in the Saccharomyces cerevisiae adhesion protein α‐agglutinin
Authors: Lipke Peter N. , Chen Min‐Hao , Nobel Hans De , Kurjan Janet , Kahn Peter C. 
Published: February 2009

AbstractThe Saccharomyces cerevisiae adhesion protein α‐agglutinin is expressed by cells of α mating type. On the basis of sequence similarities, α‐agglutinin has been proposed to contain variable‐type immunoglobulin‐like (IgV) domains. The low level of sequence similarity to IgV domains of known structure made homology modeling using standard sequence‐based alignment algorithms impossible. We have therefore developed a secondary structure‐based method that allowed homology modeling of α‐agglutinin domain III, the domain most similar to IgV domains. The model was assessed and where necessary refined to accommodate information obtained by biochemical and molecular genetic approaches, including the positions of a disulfide bond, glycosylation sites, and proteolytic sites. The model successfully predicted surface exposure of glycosylation and proteolytic sites, as well as identifying residues essential for binding activity. One side of the domain was predicted to be covered by carbohydrate residues. Surface accessibility and volume packing analyses showed that the regions of the model that have greatest sequence dissimilarity from the IgV consensus sequence are poorly structured in the biophysical sense. Nonetheless, the utility of the model suggests that these alignment and testing techniques should be of general use for building and testing of models of proteins that share limited sequence similarity with known structures.