bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biochemical characterization, localization, and tissue distribution of the longer form of mouse SIRT3
Authors: Jin Lei , Galonek Heidi , Israelian Kristine , Choy Wendy , Morrison Michael , Xia Yu , Wang Xiaohong , Xu Yihua , Yang Yuecheng , Smith Jesse J. , Hoffmann Ethan , Carney David P. , Perni Robert B. , Jirousek Michael R. , Bemis Jean E. , Milne Jill C. , Sinclair David A. , Westphal Christoph H. 
Published: July 2010

AbstractSIRT3 is a key mitochondrial protein deacetylase proposed to play key roles in regulating mitochondrial metabolism but there has been considerable debate about its actual size, the sequences required for activity, and its subcellular localization. A previously cloned mouse SIRT3 has high sequence similarity with the C‐terminus of human SIRT3 but lacks an N‐terminal mitochondrial targeting sequence and has no detectable deacetylation activity in vitro. Using 5′ rapid amplification of cDNA ends, we cloned the entire sequence of mouse SIRT3, as well as rat and rabbit SIRT3. Importantly, we find that full‐length SIRT3 protein localizes exclusively to the mitochondria, in contrast to reports of SIRT3 localization to the nucleus. We demonstrate that SIRT3 has no deacetylation activity in vitro unless the protein is truncated, consistent with human SIRT3. In addition, we determined the inhibition constants and mechanism of action for nicotinamide and a small molecule SIRT3 inhibitor against active mouse SIRT3 and show that the mechanisms are different for the two compounds with respect to peptide substrate and NAD+. Thus, identification and characterization of the actual SIRT3 sequence should help resolve the debate about the nature of mouse SIRT3 and identify new mechanisms to modulate enzymatic activity.
Gln‐41 is intermolecularly cross‐linked to Lys‐113 in F‐actin by N‐(4‐azidobenzoyl)‐putrescine
Authors: Hegyi G. , Chatterjie N. , Healy‐Louie G. , Elzinga M. , Michel H. , Shabanowitz J. , Hunt D.F. 
Published: July 2010

AbstractThe bifunctional reagent N‐(4‐azidobenzoyl)‐putrescine was synthesized and covalently bound to rabbit skeletal muscle actin. The incorporation was mediated by guinea pig liver transglutaminase under conditions similar to those described by Takashi (1988, Biochemistry 27, 938–943); up to 0.5 M/M were incorporated into G‐actin, whereas F‐actin was refractory to incorporation. Peptide fractionation showed that at least 90% of the label was bound to Gln‐41. The labeled G‐actin was polymerized, and irradiation of the F‐actin led to covalent intermo‐lecular cross‐linking. A cross‐linked peptide complex was isolated from a tryptic digest of the cross‐linked actin in which digestion was limited to arginine; sequence analysis as well as mass spectrometry indicated that the linked peptides contained residues 40–62 and residues 96–116, and that the actual cross‐link was between Gln‐41 and Lys‐113. Thus the γ‐carboxyl group of Gln‐41 must be within 10.7 Å of the side chain (probably the amino group) of Lys‐113 in an adjacent actin monomer. In the atomic model for F‐actin proposed by Holmes et al. (1990, Nature 347, 44–49), the α‐carbons of these residues in adjacent monomers along the two‐start helices are sufficiently close to permit cross‐linking of their side chains, and, pending atomic resolution of the side chains, the results presented here seem to support the proposed model.
Modeling the effects of mutations on the denatured states of proteins
Authors: Shortle David , Chan Hue Sun , Dill Ken A. 
Published: July 2010

AbstractWe develop a model for the reversible denaturation of proteins and for the effects of single‐site mutations on the denatured states. The model is based on short chains of sequences of H (hydrophobic) and P (other) monomers configured as self‐avoiding walks on the two‐dimensional square lattice. The N (native) state is defined as the unique conformation of lowest contact energy, whereas the D (denatured) state is defined as the collection of all other conformations. With this model we are able to determine the exact partition function, and thus the exact native‐denatured equilibrium for various solvent conditions, using the computer to exhaustively enumerate every possible configuration. Previous studies confirm that this model shows many aspects of protein‐like behavior. The present study attempts to model how the denatured state (1) depends on the amino acid sequence, and (2) is changed by single‐site mutations. The model accounts for two puzzling experimental results: (1) the replacement of a polar residue by a hydrophobic amino acid on the surface of a protein can destabilize a native protein, and (2) the “denaturant slope,” m = ∂δG/∂c (where c is the concentration of denaturant – urea, guanidine hydrochloride), can sometimes change by as much as 30% due to a single mutation. The principal conclusion of the present study is that, under strong folding conditions, the denatured conformations that are in equilibrium with the native state are not open random configurations. Instead, they are an ensemble of highly compact conformations with a distribution that depends on the residue sequence and that can be substantially altered by single mutations. Most importantly, we conclude that mutations can exert their dominant effects on protein stability by changing the entropy of folding.
NMR structure of oxidized Escherichia coli glutaredoxin: Comparison with reduced E. coli glutaredoxin and functionally related proteins
Authors: Xia Tai‐He , Bushweller John H. , Sodano Patrick , Billeter Martin , Björnberg Olof , Holmgren Arne , Wüthrich Kurt 
Published: July 2010

AbstractThe determination of the NMR structure of oxidized Escherichia coli glutaredoxin in aqueous solution is described, and comparisons of this structure with that of reduced E. coli glutaredoxin and the related proteins E. coli thioredoxin and T4 glutaredoxin are presented. Based on nearly complete sequence‐specific 1H‐NMR assignments, 804 nuclear Overhauser enhancement distance constraints and 74 dihedral angle constraints were obtained as the input for the structure calculations, for which the distance geometry program DIANA was used followed by simulated annealing with the program X‐PLOR. The molecular architecture of oxidized glutaredoxin is made up of three helices and a four‐stranded β‐sheet. The three‐dimensional structures of oxidized and the recently described reduced glutaredoxin are very similar. Quantitative analysis of the exchange rates of 34 slowly exchanging amide protons from corresponding series of two‐dimensional [15N, 1H]‐correlated spectra of oxidized and reduced glutaredoxin showed close agreement, indicating almost identical hydrogen‐bonding patterns. Nonetheless, differences in local dynamics involving residues near the active site and the C‐terminal α‐helix were clearly manifested. Comparison of the structure of E. coli glutaredoxin with those of T4 glutaredoxin and E. coli thioredoxin showed that all three proteins have a similar overall polypeptide fold. An area of the protein surface at the active site containing Arg 8, Cys 11, Pro 12, Tyr 13, 11e 38, Thr 58, Val 59, Pro 60, Gly 71, Tyr 72, and Thr 73 is proposed as a possible site for interaction with other proteins, in particular ribonucleotide reductase. It was found that this area corresponds to previously proposed interaction sites in T4 glutaredoxin and E. coli thioredoxin. The solvent‐accessible surface area at the active site of E. coli glutaredoxin showed a general trend to increase upon reduction. Only the sulfhydryl group of Cys 11 is exposed to the solvent, whereas that of Cys 14 is buried and solvent inaccessible.
Direct observation by X‐ray analysis of the tetrahedral “intermediate” of aspartic proteinases
Authors: Veerapandian B. , Cooper Jon B. , Šali Andrej , Blundell Tom L. , Rosati Robert L. , Dominy Beryl W. , Damon David B. , Hoover Dennis J. 
Published: July 2010

AbstractWe report the X‐ray analysis at 2.0 Å resolution for crystals of the aspartic proteinase endothiapepsin (EC 3.4.23.6) complexed with a potent difluorostatone‐containing tripeptide renin inhibitor (CP‐81,282). The scissile bond surrogate, an electrophilic ketone, is hydrated in the complex. The pro‐(R) (statine‐like) hydroxyl of the tetrahedral carbonyl hydrate is hydrogen‐bonded to both active‐site aspartates 32 and 215 in the position occupied by a water in the native enzyme. The second hydroxyl oxygen of the hydrate is hydrogen‐bonded only to the outer oxygen of Asp 32. These experimental data provide a basis for a model of the tetrahedral intermediate in aspartic proteinase‐mediated cleavage of the amide bond. This indicates a mechanism in which Asp 32 is the proton donor and Asp 215 carboxylate polarizes a bound water for nucleophilic attack. The mechanism involves a carboxylate (Asp 32) that is stabilized by extensive hydrogen bonding, rather than an oxyanion derivative of the peptide as in serine proteinase catalysis.
A functional protein hybrid between the glucose transporter and the N‐acetylglucosamine transporter of Escherichia coli
Authors: Hummel Urs , Nuoffer Claude , Zanolari Bettina , Erni Bernhard 
Published: July 2010

AbstractThe glucose and N‐acetylglucosamine‐specific transporters (IIGlc/IIIGlc and IIGlcNAc) of the bacterial phosphotransferase system mediate carbohydrate uptake across the cytoplasmic membrane concomitant with substrate phosphorylation. The two transporters have 40% amino acid sequence identity. Eight chimeric proteins between the two transporters were made by gene reconstruction. All hybrid proteins could be expressed, some inhibited cell growth, and one was active. The active hybrid transporter consists of the transmembrane domain (residues 1–386) of the IIGlc subunit and the two hydrophilic domains (residues 370–648) of IIGlcNAc. The N‐terminal hydrophilic domain of IIGlcNAc contains the transiently phosphorylated cysteine‐412. The hybrid protein is specific for glucose, which indicates that the sugar specificity determinant is in the transmembrane domain and that the cysteine from which the phosphoryl group is transferred to the substrate is not part of the binding site. The protein sequence (LKTPGRED) at which the successful fusion occurred has the characteristic properties of an interdomain oligopeptide linker (Argos, P., 1990, J. Mol. Biol. 211, 943–958).
Quantitative analysis of cyclic β‐turn models
Authors: Perczel Andraás , Fasman Gerald D. 
Published: July 2010

AbstractThe β‐turn is a frequently found structural unit in the conformation of globular proteins. Although the circular dichroism (CD) spectra of the α‐helix and β‐pleated sheet are well defined, there remains some ambiguity concerning the pure component CD spectra of the different types of β‐turns. Recently, it has been reported (Hollósi, M., Koövér, K.E., Holly, S., Radics, L., & Fasman, G.D., 1987, Biopolymers 26, 1527–1572; Perczel, A., Hollósi, M., Foxman, B.M., & Fasman, G.D., 1991a, J. Am. Chem. Soc. 113, 9772–9784) that some pseudohexapeptides (e.g., the cyclo[(δ)Ava–Gly–Pro–Aaa–Gly] where Aaa = Ser, Ser(OtBu), or Gly) in many solvents adopt a conformational mixture of type I and the type II β‐turns, although the X‐ray‐determined conformation was an ideal type I β‐turn. In addition to these pseudohexapeptides, conformational analysis was also carried out on three pseudotetrapeptides and three pseudooctapeptides. The target of the conformation analysis reported herein was to determine whether the ring stress of the above β‐turn models has an influence on their conformational properties.Quantitative nuclear Overhauser effect (NOE) measurements yielded interproton distances. The conformational average distances so obtained were interpreted utilizing molecular dynamics (MD) simulations to yield the conformational percentages. These conformational ratios were correlated with the conformational weights obtained by quantitative CD analysis of the same compounds. The pure component CD curves of type I and type II β‐turns were also obtained, using a recently developed algorithm (Perczel, A., Tusnády, G., Hollósi, M., & Fasman, G.D., 1991b, Protein Eng. 4(6), 669–679). For the first time the results of a CD deconvolution, based on the CD spectra of 14 β‐turn models, were assigned by quantitative NOE results. The NOE experiments confirmed the ratios of the component curves found for the two major β‐turns by CD analysis. These results can now be used to enhance the conformational determination of globular proteins on the basis of their CD spectra.
Selection of representative protein data sets
Authors: Hobohm Uwe , Scharf Michael , Schneider Reinhard , Sander Chris 
Published: July 2010

AbstractThe Protein Data Bank currently contains about 600 data sets of three‐dimensional protein coordinates determined by X‐ray crystallography or NMR. There is considerable redundancy in the data base, as many protein pairs are identical or very similar in sequence. However, statistical analyses of protein sequence‐structure relations require nonredundant data. We have developed two algorithms to extract from the data base representative sets of protein chains with maximum coverage and minimum redundancy. The first algorithm focuses on optimizing a particular property of the selected proteins and works by successive selection of proteins from an ordered list and exclusion of all neighbors of each selected protein. The other algorithm aims at maximizing the size of the selected set and works by successive thinning out of clusters of similar proteins. Both algorithms are generally applicable to other data bases in which criteria of similarity can be defined and relate to problems in graph theory. The largest nonredundant set extracted from the current release of the Protein Data Bank has 155 protein chains. In this set, no two proteins have sequence similarity higher than a certain cutoff (30% identical residues for aligned subsequences longer than 80 residues), yet all structurally unique protein families are represented. Periodically updated lists of representative data sets are available by electronic mail from the file server “netserv@embl‐heidelberg.de.” The selection may be useful in statistical approaches to protein folding as well as in the analysis and documentation of the known spectrum of three‐dimensional protein structures.
Dissociation of peripheral protein‐membrane complexes by high pressure
Authors: Plager Douglas A. , Nelsestuen Gary L. 
Published: July 2010

AbstractThe ability of high pressure to dissociate several peripheral protein‐membrane complexes was investigated. Three vitamin K‐dependent proteins (factor X, protein Z, and prothrombin) dissociated from small unilamellar vesicles (SUVs, 30 nm diameter) composed of 25% phosphatidylserine (PS) and 75% phosphatidylcholine (PC) at comparable pressures (midpoints of 0.3–0.6 kbar). The pressure‐induced dissociation curves for the factor X‐SUV interaction followed the expected behavior for an interaction with an apparent dissociation equilibrium constant at atmospheric pressure, KD(atm), of 9 × 10−7 M and a change in volume of association, ΔVa, of 88 mL/mol. Factor X also dissociated from large unilamellar vesicles (LUVs, 100 nm diameter, 25% PS:75% PC) with a midpoint of 0.5 kbar. A second group of calcium‐dependent membrane‐binding proteins included protein kinase C (PKC), a 64‐kDa protein, and a 32‐kDa protein. The 32‐kDa protein dissociated from SUVs (midpoint of 0.8 kbar), whereas PKC and the 64‐kDa protein did not dissociate to a significant degree. The differences in dissociability of these proteins appeared to be a result of the differences in their KD(atm)'s (decreased dissociability with decreased KD(atm)). This pattern was further demonstrated by the relatively high midpoint of dissociation (1.1–1.4 kbar) of serum amyloid P component (SAP; KD(atm) ca. 10−11) and the limited dissociation of factor Va light chain (KD(atm) ca. 10−11). Changing the vesicle composition to phosphatidylethanolamine in place of PC gave higher affinity and decreased dissociation of the 32‐kDa protein and SAP. Thus, the susceptibility of various peripheral membrane‐binding proteins to high pressure dissociation was governed by both the KD(atm) and the ΔVa of the particular protein‐membrane interaction, with the ΔVa's of the dissociable proteins being relatively small (< 150 mL/mol).
Zinc‐ and sequence‐dependent binding to nucleic acids by the N‐terminal zinc finger of the HIV‐1 nucleocapsid protein: NMR structure of the complex with the Psi‐site analog, dACGCC
Authors: South Terri L. , Summers Michael F. 
Published: July 2010

AbstractThe nucleic acid interactive properties of a synthetic peptide with sequence of the N‐terminal CCHC zinc finger (CCHC = Cys‐X2‐Cys‐X4‐His‐X4‐Cys; × = variable amino acid) of the human immunodeficiency virus (HIV) nucleocapsid protein, Zn(HIV1‐F1), have been studied by 1H NMR spectroscopy. Titration of Zn(HIV1‐F1) with oligodeoxyribonucleic acids containing different nucleotide sequences reveals, for the first time, sequence‐dependent binding that requires the presence of at least one guanosine residue for tight complex formation. The dynamics of complex formation are sensitive to the nature of the residues adjacent to guanosine, with residues on the 3′ side of guanosine having the largest influence. An oligodeoxyribonucleotide with sequence corresponding to a portion of the HIV‐1 psi‐packaging signal, d(ACGCC), forms a relatively tight complex with Zn(HIV1‐F1) (Kd = 5 × 10−6 M). Two‐dimensional nuclear Overhauser effect (NOESY) data indicate that the bound nucleic acid exists predominantly in a single‐stranded, A‐helical conformation, and the presence of more than a dozen intermolecular NOE cross peaks enabled three‐dimensional modeling of the complex. The nucleic acid binds within a hydrophobic cleft on the peptide surface. This hydrophobic cleft is defined by the side chains of residues Val1, Phe4, Ile12, and Ala13. Backbone amide protons of Phe4 and Ala13 and the backbone carbonyl oxygen of Lys2 that lie within this cleft appear to form hydrogen bonds with the guanosine O6 and N1H atoms, respectively. In addition, the positively charged side chain of Arg14 is ideally positioned for electrostatic interactions with the phosphodiester backbone of the nucleic acid. The structural findings provide a rationalization for the general conservation of these hydrophobic and basic residues in CCHC zinc fingers, and are consistent with site‐directed mutagenesis results that implicate these residues as direct participants in viral genome recognition.
Modeling α‐helical transmembrane domains: The calculation and use of substitution tables for lipid‐facing residues
Authors: Blundell Tom L. , Donnelly Dan , Overington John P. , Ruffle Stuart V. , Nugent Jonathan H.A. 
Published: July 2010

AbstractAmino acid substitution tables are calculated for residues in membrane proteins where the side chain is accessible to the lipid. The analysis is based upon the knowledge of the three‐dimensional structures of two homologous bacterial photosynthetic reaction centers and alignments of their sequences with the sequences of related proteins. The patterns of residue substitutions show that the lipid‐accessible residues are less conserved and have distinctly different substitution patterns from the inaccessible residues in water‐soluble proteins. The observed substitutions obtained from sequence alignments of transmembrane regions (identified from, e.g., hydrophobicity analysis) can be compared with the patterns derived from the substitution tables to predict the accessibility of residues to the lipid. A Fourier transform method, similar to that used for the calculation of a hydrophobic moment, is used to detect periodicity in the predicted accessibility that is compatible with the presence of an α‐helix. If the putative transmembrane region is identified as helical, then the buried and exposed faces can be discriminated. The presence of charged residues on the lipid‐exposed face can help to identify the regions that are in contact with the polar environment on the borders of the bilayer, and the construction of a meaningful three‐dimensional model is then possible. This method is tested on an alignment of bacteriorhodopsin and two related sequences for which there are structural data at near atomic resolution.
Helical peptides with three pairs of Asp‐Arg and Glu‐Arg residues in different orientations and spacings
Authors: Huyghues‐Despointes Beatrice M.P. , Martin Scholtz J. , Baldwin Robert L. 
Published: July 2010

AbstractThe helix‐stabilizing effects of repeating pairs of Asp‐Arg and Glu‐Arg residues have been characterized using a peptide system of the same design used earlier to study Glu‐Lys (Marqusee, S. & Baldwin, R.L., 1987, Proc. Natl. Acad. Sci. USA 84, 8898–8902) and Asp‐Lys ion pairs (Marqusee, S. & Baldwin, R.L., 1990, In Protein Folding [Gierasch, L.M. & King, J., Eds.], pp. 85–94, AAAS, Washington, D.C.). The consequences of breaking ion pair and charge‐helix dipole interactions by titration to pH 2 have been compared with the results of screening these interactions with NaCl at pH 7.0 and pH 2.5. The four peptides in each set contain three pairs of acidic (A) and basic (B) residues spaced either i, i + 4 or i, i + 3 apart. In one peptide of each kind the pairwise order of residues is AB, with the charges oriented favorably to the helix macrodipole, and in the other peptide the order is BA.The results are as follows: (1) Remarkably, both Asp‐Arg and Glu‐Arg peptides show the same pattern of helix stabilization at pH 7.0 found earlier for Glu‐Lys and Asp‐Lys peptides: i + 4 AB > i + 4 BA = i + 3 AB > i + 3 BA. (2) The ion pairs and charge‐helix dipole interactions cannot be cleanly separated, but the results suggest that both interactions make important contributions to helix stability. Because the four peptides of each set have nearly identical compositions, the large differences in helix content within each set cannot be caused by differences in length and sequence. (3) Significant differences in helix content remain at 5 M NaCl, where ion pair interactions are completely screened. The residual interactions may either be nonscreenable H‐bond interactions or remaining charge‐helix dipole interactions.