bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stabilization of a metastable state of Torpedo californica acetylcholinesterase by chemical chaperones
Authors: Millard Charles B. , Shnyrov Valery L. , Newstead Simon , Shin Irina , Roth Esther , Silman Israel , Weiner Lev 
Published: September 2003

AbstractChemical modification of Torpedo californica acetylcholinesterase by the natural thiosulfinate allicin produces an inactive enzyme through reaction with the buried cysteine Cys 231. Optical spectroscopy shows that the modified enzyme is “native‐like,” and inactivation can be reversed by exposure to reduced glutathione. The allicin‐modified enzyme is, however, metastable, and is converted spontaneously and irreversibly, at room temperature, with t1/2 ≃ 100 min, to a stable, partially unfolded state with the physicochemical characteristics of a molten globule. Osmolytes, including trimethylamine‐N‐oxide, glycerol, and sucrose, and the divalent cations, Ca2+, Mg2+, and Mn2+ can prevent this transition of the native‐like state for >24 h at room temperature. Trimethylamine‐N‐oxide and Mg2+ can also stabilize the native enzyme, with only slight inactivation being observed over several hours at 39°C, whereas in their absence it is totally inactivated within 5 min. The stabilizing effects of the osmolytes can be explained by their differential interaction with the native and native‐like states, resulting in a shift of equilibrium toward the native state. The stabilizing effects of the divalent cations can be ascribed to direct stabilization of the native state, as supported by differential scanning calorimetry.
The crystal structure of Escherichia coli heat shock protein YedU reveals three potential catalytic active sites
Authors: Zhao Yonghong , Liu Deqian , Kaluarachchi Warna D. , Bellamy Henry D. , White Mark A. , Fox Robert O. 
Published: September 2003

AbstractThe mRNA of Escherichia coli yedU gene is induced 31‐fold upon heat shock. The 31‐kD YedU protein, also calls Hsp31, is highly conserved in several human pathogens and has chaperone activity. We solved the crystal structure of YedU at 2.2 Å resolution. YedU monomer has an α/β/α sandwich domain and a small α/β domain. YedU is a dimer in solution, and its crystal structure indicates that a significant amount of surface area is buried upon dimerization. There is an extended hydrophobic patch that crosses the dimer interface on the surface of the protein. This hydrophobic patch is likely the substrate‐binding site responsible for the chaperone activity. The structure also reveals a potential protease‐like catalytic triad composed of Cys184, His185, and Asp213, although no enzymatic activity could be identified. YedU coordinates a metal ion using His85, His122, and Glu90. This 2‐His‐1‐carboxylate motif is present in carboxypeptidase A (a zinc enzyme), and a number of dioxygenases and hydroxylases that utilize iron as a cofactor, suggesting another potential function for YedU.
Exploring the sequence‐structure protein landscape in the glycosyltransferase family
Authors: Zhang Ziding , Kochhar Sunil , Grigorov Martin 
Published: September 2003

AbstractTo understand the molecular basis of glycosyltransferases' (GTFs) catalytic mechanism, extensive structural information is required. Here, fold recognition methods were employed to assign 3D protein shapes (folds) to the currently known GTF sequences, available in public databases such as GenBank and Swissprot. First, GTF sequences were retrieved and classified into clusters, based on sequence similarity only. Intracluster sequence similarity was chosen sufficiently high to ensure that the same fold is found within a given cluster. Then, a representative sequence from each cluster was selected to compose a subset of GTF sequences. The members of this reduced set were processed by three different fold recognition methods: 3D‐PSSM, FUGUE, and GeneFold. Finally, the results from different fold recognition methods were analyzed and compared to sequence‐similarity search methods (i.e., BLAST and PSI‐BLAST). It was established that the folds of about 70% of all currently known GTF sequences can be confidently assigned by fold recognition methods, a value which is higher than the fold identification rate based on sequence comparison alone (48% for BLAST and 64% for PSI‐BLAST). The identified folds were submitted to 3D clustering, and we found that most of the GTF sequences adopt the typical GTF A or GTF B folds. Our results indicate a lack of evidence that new GTF folds (i.e., folds other than GTF A and B) exist. Based on cases where fold identification was not possible, we suggest several sequences as the most promising targets for a structural genomics initiative focused on the GTF protein family.
Molecular dynamics simulation of highly charged proteins: Comparison of the particle‐particle particle‐mesh and reaction field methods for the calculation of electrostatic interactions
Authors: Gargallo Raimundo , Hünenberger Philippe H. , Avilés Francesc X. , Oliva Baldomero 
Published: September 2003

AbstractMolecular dynamics (MD) simulations of the activation domain of porcine procarboxypeptidase B (ADBp) were performed to examine the effect of using the particle‐particle particle‐mesh (P3M) or the reaction field (RF) method for calculating electrostatic interactions in simulations of highly charged proteins. Several structural, thermodynamic, and dynamic observables were derived from the MD trajectories, including estimated entropies and solvation free energies and essential dynamics (ED). The P3M method leads to slightly higher atomic positional fluctuations and deviations from the crystallographic structure, along with somewhat lower values of the total energy and solvation free energy. However, the ED analysis of the system leads to nearly identical results for both simulations. Because of the strong similarity between the results, both methods appear well suited for the simulation of highly charged globular proteins in explicit solvent. However, the lower computational demand of the RF method in the present implementation represents a clear advantage over the P3M method.
Binding‐induced folding transitions in calpastatin subdomains A and C
Authors: Mucsi Zoltán , Hudecz Ferenc , Hollósi Miklós , Tompa Peter , Friedrich Peter 
Published: September 2003

AbstractCalpastatin, the endogenous inhibitor of calpain, is an intrinsically unstructured protein proposed to undergo folding transitions upon binding to the enzyme. As this feature has never been experimentally tested, we have set out to characterize the conformation of two peptides corresponding to its conserved subdomains, A and C, known to interact with calpain in a Ca2+‐dependent manner. The peptides are disordered in water but show a high propensity for α‐helical conformation in the presence of trifluoroethanol. The conformational transition is sensitive to Ca2+, and is clearly seen upon binding of the peptides to the enzyme. Secondary‐structure prediction of all calpastatin sequences shows that the helix‐forming potential within these regions is a conserved feature of the inhibitor. Furthermore, quantitative data on the binding strength of calpastatin fragments reveal that binding of the inhibitor is accompanied by a large decrease in its configurational entropy. Taken together, these observations point to significant binding‐induced local folding transitions in calpastatin, in a way that ensures highly specific, yet reversible, action of the inhibitor.
On the structural and functional modularity of glycinamide ribonucleotide formyltransferases
Authors: Lee Seung‐Goo , Lutz Stefan , Benkovic Stephen J. 
Published: September 2003

AbstractGlycinamide ribonucleotide formyltransferases (GARTs) are part of the de novo purine biosynthetic pathway, catalyzing the direct transfer of a formyl group from the tetrahydrofolate cofactor to the glycinamide ribonucleotide substrate. Despite the low amino acid‐sequence identity between the GARTs from Escherichia coli and human, their tertiary structures are superimposable. As part of our functional studies of these enzymes, we have investigated the interchangeability of individual protein fragments or modules between the two enzymes and the functional properties of the resulting hybrids. The modular nature of GART facilitated the creation of combinatorial libraries of chimeras between the Escherichia coli and human enzymes, which were functionally selected through complementation of an auxotrophic Escherichia coli strain. From a pool of several dozen sequence distinct hybrids, six in vivo‐functional fusion genes were selected, overexpressed, and purified to homogeneity. The kinetic analysis of these constructs and the comparison of their kcat and KM values to the parental enzymes suggest that the characteristic kinetic properties from the two parents are “modular encoded” and can be exchanged by domain swapping. The chimeras in general, however, are subject to temperature instability and misfolding; thus, they serve primarily as useful candidates for further rounds of optimization.
Isolation and characterization of human nuclear and cytosolic multisynthetase complexes and the intracellular distribution of p43/EMAPII
Authors: Wolfe Cindy L. , Warrington J. Anthony , Davis Stanitia , Green Sherrina , Norcum Mona T. 
Published: September 2003

AbstractIn this study, the human multienzyme aminoacyl–tRNA synthetase “core” complex has been isolated from the nuclear and cytosolic compartments of human cells and purified to near homogeneity. It is clear from the polypeptide compositions, stoichiometries, and three‐dimensional structures that the cytosolic and nuclear particles are very similar to each other and to the particle obtained from rabbit reticulocytes. The most significant difference observed via aminoacylation activity assays and densitometric analysis of electrophoretic band patterns is a lower amount of glutaminyl–tRNA synthetase in the human particles. However, this is not enough to cause major changes in the three‐dimensional structures calculated from samples negatively stained with either uranyl acetate or methylamine vanadate. Indeed, the latter samples produce volumes that are highly similar to an initial structure previously calculated from a frozen hydrated sample of the rabbit multisynthetase complex. New structures in this study reveal that the three major structural domains have discrete subsections. This information is an important step toward determination of specific protein interactions and arrangements within the multisynthetase core complex and understanding of the particle's cellular function(s). Finally, gel filtration and immunoblot analysis demonstrate that a major biological role for the cytokine precursor p43 is as an integral part of the multisynthetase complex.
The role of protein stability, solubility, and net charge in amyloid fibril formation
Authors: Schmittschmitt Jason P. , Scholtz J. Martin 
Published: September 2003

AbstractRibonuclease Sa and two charge‐reversal variants can be converted into amyloid in vitro by the addition of 2,2,2‐triflouroethanol (TFE). We report here amyloid fibril formation for these proteins as a function of pH. The pH at maximal fibril formation correlates with the pH dependence of protein solubility, but not with stability, for these variants. Additionally, we show that the pH at maximal fibril formation for a number of well‐characterized proteins is near the pI, where the protein is expected to be the least soluble. This suggests that protein solubility is an important determinant of fibril formation.
Structure and disorder in the ribonuclease S‐peptide probed by NMR residual dipolar couplings
Authors: Alexandrescu Andrei T. , Kammerer Richard A. 
Published: September 2003

AbstractNMR residual dipolar couplings for the S‐peptide of ribonuclease A aligned in C8E5/n‐octanol liquid crystals are consistent with the presence of a native‐like α‐helix structure undergoing dynamic fraying. Residues 3–13, which correspond to the first α‐helix of ribonuclease A, show couplings that become more negative at low temperature and in the presence of salt, conditions which stabilize α‐helical structure in the S‐peptide. By contrast, dipolar couplings from the N and C termini of the peptide are close to zero and remain nearly invariant with changes in solution conditions. Torsion angle dynamics simulations using a gradient of dihedral restraint bounds that increase from the center to the ends of the peptide reproduce the experimentally observed sequence dependence of dipolar couplings. The magnitudes of residual dipolar couplings depend on the anisotropy of the solute. Native proteins often achieve nearly spherical shapes due to the hydrophobic effect. Embryonic partially folded structures such as the S‐peptide α‐helix have an intrinsically greater potential for anisotropy that can result in sizable residual dipolar couplings in the absence of long‐range structure.
Kinetics of the dimerization of retroviral proteases: The “fireman's grip” and dimerization
Authors: Ingr Marek , Uhlíková Tat'ána , Str̆ís̆ovský Kvido , Majerová Eva , Konvalinka Jan 
Published: September 2003

AbstractAll retroviral proteases belong to the family of aspartic proteases. They are active as homodimers, each unit contributing one catalytic aspartate to the active site dyad. An important feature of all aspartic proteases is a conserved complex scaffold of hydrogen bonds supporting the active site, called the “fireman's grip,” which involves the hydroxyl groups of two threonine (serine) residues in the active site Asp‐Thr(Ser)‐Gly triplets. It was shown previously that the fireman's grip is indispensable for the dimer stability of HIV protease. The retroviral proteases harboring Ser in their active site triplet are less active and, under natural conditions, are expressed in higher enzyme/substrate ratio than those having Asp‐Thr‐Gly triplet. To analyze whether this observation can be attributed to the different influence of Thr or Ser on dimerization, we prepared two pairs of the wild‐type and mutant proteases from HIV and myeloblastosis‐associated virus harboring either Ser or Thr in their Asp‐Thr(Ser)‐Gly triplet. The equilibrium dimerization constants differed by an order of magnitude within the relevant pairs. The proteases with Thr in their active site triplets were found to be ∼10 times more thermodynamically stable. The dimer association contributes to this difference more than does the dissociation. We propose that the fireman's grip might be important in the initial phases of dimer formation to help properly orientate the two subunits of a retroviral protease. The methyl group of threonine might contribute significantly to fixing such an intermediate conformation.
The specificity of cross‐reactivity: Promiscuous antibody binding involves specific hydrogen bonds rather than nonspecific hydrophobic stickiness
Authors: James Leo C. , Tawfik Dan S. 
Published: September 2003

AbstractProteins are renowned for their specificity of function. There is, however, accumulating evidence that many proteins, from enzymes to antibodies, are functionally promiscuous. Promiscuity is of considerable physiological importance. In the immune system, cross‐reactive or multispecific antibodies are implicated in autoimmune and allergy conditions. In most cases, however, the mechanism behind promiscuity and the relationship between specific and promiscuous activities are unknown. Are the two contradictory? Or can a protein exhibit several unrelated activities each of which is highly specific? To address these questions, we studied a multispecific IgE antibody (SPE7) elicited against a 2,4‐dinitrophenyl hapten (DNP). SPE7 is able to distinguish between closely related derivatives such as NP (nitrophenol) and DNP, yet it can also bind a number of unrelated ligands. We find that, like DNP, the cross‐reactants are themselves bound specifically—close derivatives of these cross‐reactants show very low or no binding to SPE7. It has been suggested that cross‐reactivity is simply due to “hydrophobic stickiness”, nonspecific interactions between hydrophobic ligands and binding sites. However, partitioning experiments reveal that affinity for SPE7 is unrelated to ligand hydrophobicity. These data, combined with crystal structures of SPE7 in complex with four different ligands, demonstrate that each cross‐reactant is bound specifically, forming different hydrogen bonds dependant upon its particular chemistry and the availability of complementary antibody residues. SPE7 is highly homologous to the germline antinitrophenol (NP) antibody B1–8. By comparing the sequences and binding patterns of SPE7 and B1–8, we address the relationship between affinity maturation, specificity, and cross‐reactivity.
Contribution of active site residues to the activity and thermal stability of ribonuclease Sa
Authors: Yakovlev Gennady I. , Mitkevich Vladimir A. , Shaw Kevin L. , Trevino Saul , Newsom Stephanie , Pace C. Nick , Makarov Alexander A. 
Published: September 2003

AbstractWe have used site‐specific mutagenesis to study the contribution of Glu 74 and the active site residues Gln 38, Glu 41, Glu 54, Arg 65, and His 85 to the catalytic activity and thermal stability of ribonuclease Sa. The activity of Gln38Ala is lowered by one order of magnitude, which confirms the involvement of this residue in substrate binding. In contrast, Glu41Lys had no effect on the ribonuclease Sa activity. This is surprising, because the hydrogen bond between the guanosine N1 atom and the side chain of Glu 41 is thought to be important for the guanine specificity in related ribonucleases. The activities of Glu54Gln and Arg65Ala are both lowered about 1000‐fold, and His85Gln is totally inactive, confirming the importance of these residues to the catalytic function of ribonuclease Sa. In Glu74Lys, kcat is reduced sixfold despite the fact that Glu 74 is over 15 Å from the active site. The pH dependence of kcat/KM is very similar for Glu74Lys and wild‐type RNase Sa, suggesting that this is not due to a change in the pK values of the groups involved in catalysis. Compared to wild‐type RNase Sa, the stabilities of Gln38Ala and Glu74Lys are increased, the stabilities of Glu41Lys, Glu54Gln, and Arg65Ala are decreased and the stability of His85Gln is unchanged. Thus, the active site residues in the ribonuclease Sa make different contributions to the stability.
The physical basis of microtubule structure and stability
Authors: Sept David , Baker Nathan A. , McCammon J. Andrew 
Published: September 2003

AbstractMicrotubules are cylindrical polymers found in every eukaryotic cell. They have a unique helical structure that has implications at both the cellular level, in terms of the functions they perform, and at the multicellular level, such as determining the left–right symmetry in plants. Through the combination of an atomically detailed model for a microtubule and large‐scale computational techniques for computing electrostatic interactions, we are able to explain the observed microtubule structure. On the basis of the lateral interactions between protofilaments, we have determined that B lattice is the most favorable configuration. Further, we find that these lateral bonds are significantly weaker than the longitudinal bonds along protofilaments. This explains observations of microtubule disassembly and may serve as another step toward understanding the basis for dynamic instability.
A de novo redesign of the WW domain
Authors: Kraemer‐Pecore Christina M. , Lecomte Juliette T.J. , Desjarlais John R. 
Published: September 2003

AbstractWe have used a sequence prediction algorithm and a novel sampling method to design protein sequences for the WW domain, a small β‐sheet motif. The procedure, referred to as SPANS, designs sequences to be compatible with an ensemble of closely related polypeptide backbones, mimicking the inherent flexibility of proteins. Two designed sequences (termed SPANS‐WW1 and SPANS‐WW2), using only naturally occurring l‐amino acids, were selected for study and the corresponding polypeptides were prepared in Escherichia coli. Circular dichroism data suggested that both purified polypeptides adopted secondary structure features related to those of the target without the aid of disulfide bridges or bound cofactors. The structure exhibited by SPANS‐WW2 melted cooperatively by raising the temperature of the solution. Further analysis of this polypeptide by proton nuclear magnetic resonance spectroscopy demonstrated that at 5°C, it folds into a structure closely resembling a natural WW domain. This achievement constitutes one of a small number of successful de novo protein designs through fully automated computational methods and highlights the feasibility of including backbone flexibility in the design strategy.
Profile–profile comparisons by COMPASS predict intricate homologies between protein families
Authors: Sadreyev Ruslan I. , Baker David , Grishin Nick V. 
Published: September 2003

AbstractRecently we proposed a novel method of alignment–alignment comparison, COMPASS (the tool for COmparison of Multiple Protein Alignments with Assessment of Statistical Significance). Here we present several examples of the relations between PFAM protein families that were detected by COMPASS and that lead to the predictions of presently unresolved protein structures. We discuss relatively straightforward COMPASS predictions that are new and interesting to us, and that would require a substantial time and effort to justify even for a skilled PSI‐BLAST user. All of the presented COMPASS hits are independently confirmed by other methods, including the ab initio structure‐prediction method ROSETTA. The tertiary structure predictions made by ROSETTA proved to be useful for improving sequence‐derived alignments, because they are based on a reasonable folding of the polypeptide chain rather than on the information from sequence databases. The ability of COMPASS to predict new relations within the PFAM database indicates the high sensitivity of COMPASS searches and substantiates its potential value for the discovery of previously unknown similarities between protein families.
Association and dissociation kinetics of colicin E3 and immunity protein 3: Convergence of theory and experiment
Authors: Zhou Huan‐Xiang 
Published: September 2003

AbstractThe rapid binding of cytotoxic colicin E3 by its cognate immunity protein Im3 is essential in safeguarding the producing cell. The X‐ray structure of the E3/Im3 complex shows that the Im3 molecule interfaces with both the C‐terminal ribonuclease (RNase) domain and the N‐terminal translocation domain of E3. The association and dissociation rates of the RNase domain and Im3 show drastically different sensitivities to ionic strength, as previously rationalized for electrostatically enhanced diffusion‐limited protein–protein associations. Relative to binding to the RNase domain, binding to full‐length E3 shows a comparable association rate but a significantly lower dissociation rate. This outcome is just what was anticipated by a theory for the binding of two linked domains to a protein. The E3/Im3 system thus provides a powerful paradigm for the interplay of theory and experiment.