bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rapid amide proton exchange rates in peptides and proteins measured by solvent quenching and two‐dimensional NMR
Authors: Zhang Yu‐Zhu , Roder Heinrich , Paterson Yvonne 
Published: June 2010

AbstractIn an effort to develop a more versatile quenched hydrogen exchange method for studies of peptide conformation and protein‐ligand interactions, the mechanism of amide proton exchange for model peptides in DMSO‐D2O mixtures was investigated by NMR methods. As in water, H‐D exchange rates in the presence of 90% or 95% DMSO exhibit characteristic acid‐ and base‐catalyzed processes and negligible water catalysis. However, the base‐catalyzed rate is suppressed by as much as four orders of magnitude in 95% DMSO. As a result, the pH at which the exchange rate goes through a minimum is shifted up by about two pH units and the minimum exchange rate is ∼ 100‐fold reduced relative to that in D2O. The solvent‐dependent decrease in base‐catalyzed exchange rates can be attributed primarily to a large increase in pKa values for the NH group, whereas solvent effects on pKw seem less important. Addition of toluene and cyclohexane resulted in improved proton NMR chemical shift dispersion. The dramatic reduction in exchange rates observed in the solvent mixture at optimal pH makes it possible to apply 2D NMR for NH exchange measurements on peptides under conditions where rates are too rapid for direct NMR analysis. To test this solvent‐quenching method, melittin was exchanged in D2O (pH 3.2, 12 °C), aliquots were quenched by rapid freezing, lyophilized, and dissolved in quenching buffer (70% DMSO, 25% toluene, 4% D2O, 1% cyclohexane, 75 mM dichloroacetic acid) for NMR analysis. Exchange rates for 21 amide protons were measured by recording 2D NMR spectra on a series of samples quenched at different times. The results are consistent with a monomeric unfolded conformation of melittin at acidic pH. The ability to trap labile protons by solvent quenching makes it possible to extend amide protection studies to peptide ligands or labile protons on the surface of a protein involved in macromolecular interactions.
The metzincins — Topological and sequential relations between the astacins, adamalysins, serralysins, and matrixins (collagenases) define a super family of zinc‐peptidases
Authors: Stöcker Walter , Grams Frank , Reinemer Peter , Bode Wolfram , Baumann Ulrich , Gomis‐Rüth Franz‐Xaver , Mckay David B. 
Published: June 2010

AbstractThe three‐dimensional structures of the zinc endopeptidases human neutrophil collagenase, adamalysin II from rattle snake venom, alkaline proteinase from Pseudomonas aeruginosa, and astacin from crayfish are topologically similar, with respect to a five‐stranded β‐sheet and three α‐helices arranged in typical sequential order. The four proteins exhibit the characteristic consensus motif HEXXHXXGXXH, whose three histidine residues are involved in binding of the catalytically essential zinc ion. Moreover, they all share a conserved methionine residue beneath the active site metal as part of a superimposable “Met‐turn.” This structural relationship is supported by a sequence alignment performed on the basis of topological equivalence showing faint but distinct sequential similarity. The alkaline proteinase is about equally distant (26% sequence identity) to both human neutrophil collagenase and astacin and a little further away from adamalysin II (17% identity). The pairs astacin/adamalysin II, astacin/human neutrophil collagenase, and adamalysin II/human neutrophil collagenase exhibit sequence identities of 16%, 14%, and 13%, respectively. Therefore, the corresponding four distinct families of zinc peptidases, the astacins, the matrix metalloproteinases (matrixins, collagenases), the adamalysins/reprolysins (snake venom proteinases/reproductive tract proteins), and the serralysins (large bacterial proteases from Serratia, Erwinia, and Pseudomonas) appear to have originated by divergent evolution from a common ancestor and form a superfamily of proteolytic enzymes for which the designation “metzincins” has been proposed. There is also a faint but significant structural relationship of the metzincins to the thermolysin‐like enzymes, which share the truncated zinc‐binding motif HEXXH and, moreover, similar topologies in their N‐terminal domains.
Structural diversity in a conserved cholera toxin epitope involved in ganglioside binding
Authors: Shoham Menachem , Scherf Tali , Anglister Jacob , Levitt Michael , Merritt Ethan A. , Hol Wim G.J. 
Published: June 2010

AbstractCholera is a widespread disease for which there is no efficient vaccine. A better understanding of the conformational rearrangements at the epitope might be very helpful for the development of a good vaccine. Cholera toxin (CT) as well as the closely related heat‐labile toxin from Escherichia coli (LT) are composed of two subunits, A and B, which form an oligomeric assembly AB5. Residues 50–64 on the surface of the B subunits comprise a conserved loop (CTP3), which is involved in saccharide binding to the receptor on epithelial cells. This loop exhibits remarkable conformational plasticity induced by environmental constraints. The crystal structure of this loop is compared in the free and receptor‐bound toxins as well as in the crystal and solution structures of a complex with TE33, a monoclonal antibody elicited against CTP3. In the toxins this loop forms an irregular structure connecting a β‐strand to the central α‐helix. Ser 55 and Gin 56 exhibit considerable conformational variability in the five subunits of the unliganded toxins. Saccharide binding induces a change primarily in Ser 55 and Gin 56 to a conformation identical in all five copies. Thus, saccharide binding confers rigidity upon the loop. The conformation of CTP3 in complex with TE33 is quite different. The amino‐terminal part of CTP3 forms a β‐turn that fits snugly into a deep binding pocket on TE33, in both the crystal and NMR‐derived solution structure. Only 8 and 12 residues out of 15 are seen in the NMR and crystal structures, respectively. Despite these conformational differences, TE33 is cross‐reactive with intact CT, albeit with a thousandfold decrease in affinity. This suggests a different interaction of TE33 with intact CT.
Continuous and discontinuous domains: An algorithm for the automatic generation of reliable protein domain definitions
Authors: Siddiqui Asim S. , Barton Geoffrey J. 
Published: June 2010

AbstractAn algorithm is presented for the fast and accurate definition of protein structural domains from coordinate data without prior knowledge of the number or type of domains. The algorithm explicitly locates domains that comprise one or two continuous segments of protein chain. Domains that include more than two segments are also located.The algorithm was applied to a nonredundant database of 230 protein structures and the results compared to domain definitions obtained from the literature, or by inspection of the coordinates on molecular graphics. For 70% of the proteins, the derived domains agree with the reference definitions, 18% show minor differences and only 12% (28 proteins) show very different definitions. Three screens were applied to identify the derived domains least likely to agree with the subjective definition set. These screens revealed a set of 173 proteins, 97% of which agree well with the subjective definitions.The algorithm represents a practical domain identification tool that can be run routinely on the entire structural database. Adjustment of parameters also allows smaller compact units to be identified in proteins.
On the lack of coordination between protein folding and flavin insertion in Escherichia coli for flavocytochrome b2 mutant forms Y254L and D282N
Authors: Gondry Muriel , Diêp Lê K.H. , Lederer Florence , Manson Forbes D.C. , Reid Graeme A. , Chapman Stephen K. , Scott Mathews F. 
Published: June 2010

AbstractWild‐type flavocytochrome b2 (L‐lactate dehydrogenase) from Saccharotnyces cerevisiae, as well as a number of its point mutants, can be expressed to a reasonable level as recombinant proteins in Escherichia coli (20–25 mg per liter culture) with a full complement of prosthetic groups. At the same expression level, active‐site mutants Y254L and D282N, on the other hand, were obtained with an FMN/heme ratio significantly less than unity, which could not be raised by addition of free FMN. Evidence is provided that the flavin deficit is due to incomplete prosthetic group incorporation during biosynthesis. Flavin‐free and holo‐forms for both mutants could be separated on a Blue‐Trisacryl M column. The far‐UV CD spectra of the two forms of each mutant protein were very similar to one another and to that of the wild‐type enzyme, suggesting the existence of only local conformational differences between the active holo‐enzymes and the nonreconstitutable flavin‐free forms. Selective proteolysis with chymotrypsin attacked the same bond for the two mutant holo‐enzymes as in the wild‐type one, in the protease‐sensitive loop. In contrast, for the flavin‐free forms of both mutants, cleavage occurred at more than a single bond. Identification of the cleaved bonds suggested that the structural differences between the mutant flavin‐free and holo‐forms are located mostly at the C‐terminal end of the barrel, which carries the prosthetic group and the active site. Altogether, these findings suggest that the two mutations induce an alteration of the protein‐folding process during biosynthesis in E. coli; as a result, the synchrony between folding and flavin insertion is lost. Finally, a preliminary kinetic characterization of the mutant holo‐forms showed the Km value for lactate to be little affected; kcat, values fell by a factor of about 70 for the D282N mutant and of more than 500 for the Y254L mutant, compared to the wild‐type enzyme.
Investigation of a side‐chain‐side‐chain hydrogen bond by mutagenesis, thermodynamics, and NMR spectroscopy
Authors: Hammen Philip K. , Klevit Rachel E. , Martin Scholtz J. , William Anderson J. , Bruce Waygood E. 
Published: June 2010

AbstractAnomalous NMR behavior of the hydroxyl proton resonance for Ser 31 has been reported for histidine‐containing protein (HPr) from two microorganisms: Escherichia coli and Staphylococcus aureus. The unusual slow exchange and chemical shift exhibited by the resonance led to the proposal that the hydroxyl group is involved in a strong hydrogen bond. To test this hypothesis and to characterize the importance of such an interaction, a mutant in which Ser 31 is replaced by an alanine was generated in HPr from Escherichia coli. The activity, stability, and structure of the mutant HPr were assessed using a reconstituted assay system, analysis of solvent denaturation curves, and NMR, respectively. Substitution of Ser 31 yields a fully functional protein that is only slightly less stable (ΔΔGfolding = 0.46 ± 0.15 kcal mor−1) than the wild type. The NMR results confirm the identity of the hydrogen bond acceptor as Asp 69 and reveal that it exists as the gauche− conformer in wild‐type HPr in solution but exhibits conformational averaging in the mutant protein. The side chain of Asp 69 interacts with two main‐chain amide protons in addition to its interaction with the side chain of Ser 31 in the wild‐type protein. These results indicate that removal of the serine has led to the loss of all three hydrogen bond interactions involving Asp 69, suggesting a cooperative network of interactions. A complete analysis of the thermodynamics was performed in which differences in side‐chain hydrophobicity and conformational entropy between the two proteins are accounted for. This analysis, performed in the context of information afforded by the NMR studies, indicates that this network of interactions contributes ca. 4–5 kcal mol−1 to the conformational free energy of wild‐type HPr.
Covalent control of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase: Insights into autoregulation of a bifunctional enzyme
Authors: Kurland Irwin J. , Pilkis Simon J. 
Published: June 2010

AbstractThe hepatic bifunctional enzyme, 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (6PF‐2‐K/Fru‐2,6‐P2ase), E.C. 2–7–1–105/E.C. 3–1–3–46, is one member of a family of unique bifunctional proteins that catalyze the synthesis and degradation of the regulatory metabolite fructose‐2,6‐bisphosphate (Fru‐2,6‐P2). Fru‐2,6‐P2 is a potent activator of the glycolytic enzyme 6‐phosphofructo‐1‐kinase and an inhibitor of the gluconeogenic enzyme fructose‐1,6‐bisphosphatase, and provides a switching mechanism between these two opposing pathways of hepatic carbohydrate metabolism. The activities of the hepatic 6PF‐2‐K/Fru‐2,6‐P2ase isoform are reciprocally regulated by a cyclic AMP‐dependent protein kinase (cAPK)‐catalyzed phosphorylation at a single NH2‐terminal residue, Ser‐32. Phosphorylation at Ser‐32 inhibits the kinase and activates the bisphosphatase, in part through an electrostatic mechanism. Substitution of Asp for Ser‐32 mimics the effects of cAPK‐catalyzed phosphorylation. In the dephosphorylated homodimer, the NH2‐ and COOH‐terminal tail regions also have an interaction with their respective active sites on the same subunit to produce an autoregulatory inhibition of the bisphosphatase and activation of the kinase. In support of this hypothesis, deletion of either the NH2‐ or COOH‐terminal tail region, or both regions, leads to a disruption of these interactions with a maximal activation of the bisphosphatase. Inhibition of the kinase is observed with the NH2‐truncated forms, in which there is also a diminution of cAPK phosphorylation to decrease the Km for Fru‐6‐P. Phosphorylation of the bifunctional enzyme by cAPK disrupts these autoregulatory interactions, resulting in inhibition of the kinase and activation of the bisphosphatase. Therefore, effects of cyclic AMP‐dependent phosphorylation are mediated by a combination of electrostatic and autoregulatory control mechanisms.
Quantitative approaches to utilizing mutational analysis and disulfide crosslinking for modeling a transmembrane domain
Authors: Lee Geoffrey F. , Hazelbauer Gerald L. 
Published: June 2010

AbstractThe transmembrane domain of chemoreceptor Trg from Escherichia coli contains four transmembrane segments in its native homodimer, two from each subunit. We had previously used mutational analysis and sulfhydryl cross‐linking between introduced cysteines to obtain data relevant to the three‐dimensional organization of this domain. In the current study we used Fourier analysis to assess these data quantitatively for periodicity along the sequences of the segments. The analyses provided a strong indication of α‐helical periodicity in the first transmembrane segment and a substantial indication of that periodicity for the second segment. On this basis, we considered both segments as idealized α‐helices and proceeded to model the transmembrane domain as a unit of four helices. For this modeling, we calculated helical crosslinking moments, parameters analogous to helical hydrophobic moments, as a quantitative way of condensing and utilizing a large body of crosslinking data. Crosslinking moments were used to define the relative separation and orientation of helical pairs, thus creating a quantitatively derived model for the transmembrane domain of Trg. Utilization of Fourier transforms to provide a quantitative indication of periodicity in data from analyses of transmembrane segments, in combination with helical crosslinking moments to position helical pairs should be useful in modeling other transmembrane domains.
A phage display system for studying the sequence determinants of protein folding
Authors: Gu Hongdi , Yi Qian , Bray Susan T. , Riddle David S. , Baker David , Shiau Andrew K. 
Published: June 2010

AbstractWe have developed a phage display system that provides a means to select variants of the IgG binding domain of peptostreptococcal protein L that fold from large combinatorial libraries. The premise underlying the selection scheme is that binding of protein L to IgG requires that the protein be properly folded. Using a combination of molecular biological and biophysical methods, we show that this assumption is valid. First, the phage selection procedure strongly selects against a point mutation in protein L that disrupts folding but is not in the IgG binding interface. Second, variants recovered from a library in which the first third of protein L was randomized are properly folded. The degree of sequence variation in the selected population is striking: the variants have as many as nine substitutions in the 14 residues that were mutagenized. The approach provides a selection for “foldedness” that is potentially applicable to any small binding protein.
Incorporation of pairwise interactions into the Lifson‐Roig model for helix prediction
Authors: Shalongo William , Stellwagen Earle 
Published: June 2010

AbstractThe helix/coil equilibrium of a peptide in solution can be modulated by a variety of side‐chain interactions that are not incorporated into the standard statistical mechanical models for prediction of peptide helical content. In this report, we describe a recursive formulation of the Lifson‐Roig model that facilitates incorporation of specific pairwise side‐chain interactions as well as nonspecific individual side‐chain capping interactions. Application of this extended model to a series of host/guest peptides indicates that the apparent AG value for a pairwise apolar interaction is dependent upon the spacing and orientation but not the sequential location of the participating residues. The apparent AG values for such interactions are about 40% greater than the corresponding apparent ΔΔG values obtained from difference measurements.
Domains in folding of model proteins
Authors: Abkevich V.I. , Gutin A.M. , Shakhnovich E.I. 
Published: June 2010

AbstractBy means of Monte Carlo simulation, we investigated the equilibrium between folded and unfolded states of lattice model proteins. The amino acid sequences were designed to have pronounced energy minimum target conformations of different length and shape. For short fully compact (36‐mer) proteins, the all‐or‐none transition from the unfolded state to the native state was observed. This was not always the case for longer proteins. Among 12 designed sequences with the native structure of a fully compact 48‐mer, a simple all‐or‐none transition was observed in only three cases. For the other nine sequences, three states of behavior — the native, denatured, and intermediate states — were found. The contiguous part of the native structure (domain) was conserved in the intermediate state, whereas the remaining part was completely unfolded and structureless. These parts melted separately from each other.
Fuzzy cluster analysis of simple physicochemical properties of amino acids for recognizing secondary structure in proteins
Authors: Mocz Gabor 
Published: June 2010

AbstractFuzzy cluster analysis has been applied to the 20 amino acids by using 65 physicochemical properties as a basis for classification. The clustering products, the fuzzy sets (i.e., classical sets with associated membership functions), have provided a new measure of amino acid similarities for use in protein folding studies. This work demonstrates that fuzzy sets of simple molecular attributes, when assigned to amino acid residues in a protein's sequence, can predict the secondary structure of the sequence with reasonable accuracy. An approach is presented for discriminating standard folding states, using near‐optimum information splitting in half‐overlapping segments of the sequence of assigned membership functions. The method is applied to a nonredundant set of 252 proteins and yields ˜73% matching for correctly predicted and correctly rejected residues with ˜60% overall success rate for the correctly recognized ones in three folding states: α‐helix, β‐strand, and coil. The most useful attributes for discriminating these states appear to be related to size, polarity, and thermodynamic factors. Van der Waals volume, apparent average thickness of surrounding molecular free volume, and a measure of dimensionless surface electron density can explain ˜95% of prediction results. Hydrogen bonding and hydrophobicity indices do not yet enable clear clustering and prediction.
De novo prediction of polypeptide conformations using dihedral probability grid Monte Carlo methodology
Authors: Evans John Spencer , Chan Sunney I. , Mathiowetz Alan M. , Goddard William A. 
Published: June 2010

AbstractWe tested the dihedral probability grid Monte Carlo (DPG‐MC) methodology to determine optimal conformations of polypeptides by applying it to predict the low energy ensemble for two peptides whose solution NMR structures are known: integrin receptor peptide (YGRGDSP, Type II β‐turn) and S3 α‐helical peptide (YMSEDELKAAEAAFKRHGPT).DPG‐MC involves importance sampling, local random stepping in the vicinity of a current local minima, and Metropolis sampling criteria for acceptance or rejection of new structures. Internal coordinate values are based on side‐chain‐specific dihedral angle probability distributions (from analysis of high‐resolution protein crystal structures). Important features of DPG‐MC are: (1) Each DPG‐MC step selects the torsion angles (ϕ, ψ, χ) from a discrete grid that are then applied directly to the structure. The torsion angle increments can be taken as S = 60, 30, 15, 10, or 5°, depending on the application. (2) DPG‐MC utilizes a temperature‐dependent probability function (P) in conjunction with Metropolis sampling to accept or reject new structures.For each peptide, we found close agreement with the known structure for the low energy conformational ensemble located with DPG‐MC. This suggests that DPG‐MC will be useful for predicting conformations of other polypeptides.
A putative FAD‐binding domain in a distinct group of oxidases including a protein involved in plant development
Authors: Mushegian Arcady R. , Koonin Eugene V. 
Published: June 2010

AbstractUsing methods for database screening with individual protein sequences and alignment blocks, a conserved domain is delineated in a group of proteins including several FAD‐dependent oxidases. Two motifs within this domain resemble phosphate‐binding loops and may be directly involved in FAD binding. These motifs can be readily distinguished from previously described nucleotide‐binding sites using a method for database screening with position‐dependent weight matrices derived from alignment blocks. Unexpectedly, this group of known and predicted FAD‐dependent oxidases includes the product of the DIMINUTO gene, which is involved in Arabidopsis development, and its homologues from man and Mycobacterium leprae.
Explicit solvent dynamics and energetics of HIV‐1 protease flap opening and closing
Authors: Sadiq S. Kashif , De Fabritiis Gianni 
Published: June 2010

AbstractAn accurate description of the conformational dynamics of the β‐hairpin flaps of HIV‐1 protease is of central importance in elucidating the functional recognition of the enzyme by ligands. Using all‐atom molecular dynamics simulations in explicit solvent, with a total of 461 trajectories of ∼50 ns each, we report the closed, semiopen, open, and wide‐open flap conformation of the free wild‐type protease. The free energy of flap opening and closing from the semiopen state is 0.9 ± 0.2 and 2.4 ± 0.4 kcal/mol, respectively. The mean relaxation time of opening is ∼8 ns, in good agreement with NMR data. The explicit solvent simulations quantitatively confirm the hypothesis that the semiopen state is the dominant population in the free protease whilst fast flap tip fluctuations lead frequently to an open state. More pronounced flap rearrangements lead to a rare wide‐open state with the catalytic site completely exposed to the solvent. The structures of the different flap conformations provided herein are of general interest for improved drug design of HIV‐1 protease, in particular, the wide‐open conformation could be favored by the large Gag and GagPol polyprotein chains. Strategies that take into account multiple flap‐gating mechanisms may lead to more effective inhibitors. Proteins 2010. © 2010 Wiley‐Liss, Inc.