bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sequence analysis of the AAA protein family
Authors: Beyer Andreas 
Published: February 2009

AbstractThe AAA protein family, a recently recognized group of Walker‐type ATPases, has been subjected to an extensive sequence analysis. Multiple sequence alignments revealed the existence of a region of sequence similarity, the so‐called AAA cassette. The borders of this cassette were localized and within it, three boxes of a high degree of conservation were identified. Two of these boxes could be assigned to substantial parts of the ATP binding site (namely, to Walker motifs A and B); the third may be a portion of the catalytic center. Phylogenetic trees were calculated to obtain insights into the evolutionary history of the family. Subfamilies with varying degrees of intra‐relatedness could be discriminated; these relationships are also supported by analysis of sequences outside the canonical AAA boxes: within the cassette are regions that are strongly conserved within each subfamily, whereas little or even no similarity between different subfamilies can be observed. These regions are well suited to define fingerprints for subfamilies. A secondary structure prediction utilizing all available sequence information was performed and the result was fitted to the general 3D structure of a Walker A/GTPase. The agreement was unexpectedly high and strongly supports the conclusion that the AAA family belongs to the Walker superfamily of A/GTPases.
Ideal architecture of residue packing and its observation in protein structures
Authors: Raghunathan G. , Jernigan R. L. 
Published: February 2009

AbstractA simple model of sphere packing has been investigated as an ideal model for long‐range interactions for the packing of non‐bonded residues in protein structures. By superposing all residues, the geometry of packing around a central residue is investigated. It is found that all residues conform almost perfectly to this lattice model for sphere packing when a radius of 6.5 Å is used to define non‐bonded (virtual) interacting residues. Side‐chain positions with respect to sequential backbone segments are relatively regular as well. This lattice can readily be used in conformation simulations to reduce the conformational space.
The structures of thymidine kinase fromHerpes simplexvirus type 1 in complex with substrates and a substrate analogue
Authors: Wild Klemens , Bohner Thomas , Folkers Gerd , Schulz Georg E. 
Published: February 2009

AbstractThymidine kinase fromHerpes simplexvirus type 1 (TK) was crystallized in an N‐terminally truncated but fully active form. The structures of TK complexed with ADP at the ATP‐site and deoxythymidine‐5′‐monophosphate (dTMP), deoxythymidine (dT), or idoxuridine‐5′‐phosphate (5‐iodo‐dUMP) at the substrate‐site were refined to 2.75 Å, 2.8 Å, and 3.0 Å resolution, respectively. TK catalyzes the phosphorylation of dT resulting in an ester, and the phosphorylation of dTMP giving rise to an anhydride. The presented TK structures indicate that there are only small differences between these two modes of action. Glu83 serves as a general base in the ester reaction. Arg163 parks at an internal aspartate during ester formation and binds the α‐phosphate of dTMP during anhydride formation. The bound deoxythymidine leaves a 35 Å3cavity at position 5 of the base and two sequestered water molecules at position 2. Cavity and water molecules reduce the substrate specificity to such an extent that TK can phosphorylate various substrate analogues useful in pharmaceutical applications. TK is structurally homologous to the well‐known nucleoside monophosphate kinases but contains large additional peptide segments.
Phosphorylation on histidine is accompanied by localized structural changes in the phosphocarrier protein, HPr from Bacillus subtilis
Authors: Jones Bryan E. , Rajagopal Ponni , Klevit Rachel E. 
Published: February 2009

AbstractThe histidine‐containing protein (HPr) of bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) serves a central role in a series of phosphotransfer reactions used for the translocation of sugars across cell membranes. These studies report the high‐definition solution structures of both the unphosphorylated and histidine phosphorylated (P‐His) forms of HPr from Bacillus subtilis. Consistent with previous NMR studies, local conformational adjustments occur upon phosphorylation of His 15, which positions the phosphate group to serve as a hydrogen bond acceptor for the amide protons of Ala 16 and Arg 17 and to interact favorably with the α‐helix macrodipole. However, the positively charged side chain of the highly conserved Arg 17 does not appear to interact directly with phospho‐His 15, suggesting that Arg 17 plays a role in the recognition of other PTS enzymes or in phosphotransfer reactions directly. Unlike the results reported for Escherichia coli P‐His HPr (Van Nuland NA, Boelens R, Scheek RM, Robillard GT, 1995, J Mol Biol 246:180‐193), our data indicate that phosphorylation of His 15 is not accompanied by adoption of unfavorable backbone conformations for active site residues in B. subtilis P‐Ser HPr.
Identification and characterization of glycosylation sites in human serum clusterin
Authors: Kapron James T. , Hilliard George M. , Lakins Johnathon N. , Tenniswood MARTIN P. R. , West Karen A. , Carr Steven A. , Crabb John W. 
Published: February 2009

AbstractClusterin is a ubiquitous, heterodimeric glycoprotein with multiple possible functions that are likely influenced by glycosylation. Identification of oligosaccharide attachment sites and structural characterization of oligosaccharides in human serum clusterin has been performed by mass spectrometry and Edman degradation. Matrix‐assisted laser desorption ionization mass spectrometry revealed two molecular weight species of holoclusterin (58,505 + 250 and 63,507 + 200). Mass spectrometry also revealed molecular heterogeneity associated with both the α and β subunits of clusterin, consistent with the presence of multiple glycoforms. The data indicate that clusterin contains 17‐27% carbohydrate by weight, the α subunit contains 0‐30% carbohydrate and the β subunit contains 27‐30% carbohydrate. Liquid chromatography electrospray mass spectrometry with stepped collision energy scanning was used to selectively identify and preparatively fractionate tryptic glycopeptides. Edman sequence analysis was then used to confirm the identities of the glycopeptides and to define the attachment sites within each peptide. A total of six N‐linked glycosylation sites were identified, three in the α subunit (α4N, αy81N, α123N) and three in the β subunit (β64N, β127N, and β147N). Seven different possible types of oligosaccharide structures were identified by mass including: a monosialo‐biantennary structure, bisialobiantennary structures without or with one fucose, trisialotriantennary structures without or with one fucose, and possibly a trisialotriantennary structure with two fucose and/or a tetrasialotriantennary structure. Site β64N exhibited the least glycosylation diversity, with two detected types of oligosaccharides, and site β147N exhibited the greatest diversity, with five or six detected types of oligosaccharides. Overall, the most abundant glycoforms detected were bisialobiantennary without fucose and the least abundant were monosialobiantennary, trisialotriantennay with two fucose and/or tetrasialotriantennary. Clusterin peptides accounting for 99% of the primary structure were identified from analysis of the isolated α and β subunits, including all Ser‐ and Thr‐containing peptides. No evidence was found for the presence of O‐linked or sulfated oligosaccharides. The results provide a molecular basis for developing a better understanding of clusterin structure‐function relationships and the role clusterin glycosylation plays in physiological function.
Structural heterogeneity of the various forms of apomyoglobin; Implications for protein folding
Authors: Gilmanshin Rudolf , Dyer R. Brian , Callender Robert H. 
Published: February 2009

AbstractTemperature‐induced denaturation transitions of different structural forms of apomyoglobin were studied monitoring intrinsic tryptophan fluorescence. It was found that the tryptophans are effectively screened from solvent both in native and acid forms throughout most of the temperature range tested. Thus, the tryptophans' surroundings do not show a considerable change in structure where major protein conformational transitions have been found in apomyoglobin using other techniques. At high temperatures and under strong destabilizing conditions, the tryptophans' fluorescence parameters show sigmoidal thermal denaturation. These results, combined with previous studies, show that the structure of this protein is heterogeneous, including native‐like (tightly packed) and molten globule‐like substructures that exhibit conformation (denaturation) transitions under different conditions of pH and temperature (and denaturants). The results suggest that the folding of this protein proceeds via two “nucleation” events whereby native‐like contacts are formed. One of these events, which involves AGH “core” formation, appears to occur very early in the folding process, even before significant hydrophobic collapse in the rest of the protein molecule. From the current studies and other results, a rather detailed picture of the folding of myoglobin is presented, on the level of specific structures and their thermo‐dynamical properties as well as formation kinetics.
Glutathione S‐transferase can be used as a C‐terminal, enzymatically active dimerization module for a recombinant protease inhibitor, and functionally secreted into the periplasm of Escherichia coli
Authors: Tudyka Tatjana , Skerra Arne 
Published: February 2009

AbstractGlutathione S‐transferase (GST) from Schistosoma japonicum, which is widely used for the production of fusion proteins in the cytoplasm of Escherichia coli, was employed as a functional fusion module that effects dimer formation of a recombinant protein and confers enzymatic reporter activity at the same time. For this purpose GST was linked via a flexible spacer to the C‐terminus of the thiol‐protease inhibitor cystatin, whose binding properties for papain were to be studied. The fusion protein was secreted into the bacterial periplasm by means of the OmpA signal peptide to ensure formation of the two disulfide bonds in cystatin. The formation of wrong crosslinks in the oxidizing milieu was prevented by replacing three of the four exposed cysteine residues in GST. Using the tetracycline promoter for tightly controlled gene expression the soluble fusion protein could be isolated from the periplasmic protein fraction. Purification to homogeneity was achieved in one step by means of an affinity column with glutathione agarose. Alternatively, the protein was isolated via streptavidin affinity chromatography after the Strep‐tag had been appended to its C‐terminus. The GST moiety of the fusion protein was enzymatically active and the kinetic parameters were determined using glutathione and 1‐chloro‐2,4‐dinitrobenzene as substrates. Furthermore, strong binding activity for papain was detected in an ELISA. The signal with the cystatin‐GST fusion protein was much higher than with cystatin itself, demonstrating an avidity effect due to the dimer formation of GST. The quaternary structure was further confirmed by chemical crosslinking, which resulted in a specific reaction product with twice the molecular size. Thus, engineered GST is suitable as a moderately sized, secretion‐competent fusion partner that can confer bivalency to a protein of interest and promote detection of binding interactions even in cases of low affinity.
DSC studies of the conformational stability of barstar wild‐type
Authors: Schöppe Arnulf , Hinz Hans‐Jürgen , Agashe Vishwas R. , Ramachandran S. , Udgaonkar Jayant B. 
Published: February 2009

AbstractThe temperature induced unfolding of barstar wild‐type of bacillus amyloliquefaciens (90 residues) has been characterized by differential scanning microcalorimetry. The process has been found to be reversible in the pH range from 6.4 to 8.3 in the absence of oxygen. It has been clearly shown by a ratio of δHvH/δHcal near 1 that denaturation follows a two‐state mechanism. For comparison, the C82A mutant was also studied. This mutant exhibits similar reversibility, but has a slightly lower transition temperature. The transition enthalpy of barstar wt (303 kJ mol−1) exceeds that of the C82A mutant (276 kJ mol−1) by approximately 10%. The heat capacity changes show a similar difference, δCp being 5.3 + 1 kJ mol−1 K−1 for the wild‐type and 3.6 δ 1 kJ mol−1 K−1 for the C82A mutant. The extrapolated stability parameters at 25 °C are δG° = 23.5 + 2 kJ mol−1 for barstar wt and δG0 = 25.5 + 2 kJ mol−1 for the C82A mutant.
Human recombinant [C22A] FK506‐binding protein amide hydrogen exchange rates from mass spectrometry match and extend those from NMR
Authors: Zhang Zhongqi , Li Weiqun , Logan Timothy M. , Li Ming , Marshall Alan G. 
Published: February 2009

AbstractHydrogen/deuterium exchange behavior of human recombinant [C22A] FK506 binding protein (C22A FKBP) has been determined by protein fragmentation, combined with electrospray Fourier transform ion cyclotron resonance mass spectrometry (MS). After a specified period of H/D exchange in solution, C22A FKBP was digested by pepsin under slow exchange conditions (pH 2.4, 0°C), and then subjected to on‐line HPLC/MS for deuterium analysis of each proteolytic peptide. The hydrogen exchange rate of each individual amide hydrogen was then determined independently by heteronuclear two‐dimensional NMR on 15N‐enriched C22A FKBP. A maximum entropy method (MEM) algorithm makes it possible to derive the distributions of hydrogen exchange rate constants from the MS‐determined deuterium exchange‐in curves in either the holoprotein or its proteolytic segments. The MEM‐derived rate constant distributions of C22A FKBP and different segments of C22A FKBP are compared to the rate constants determined by NMR for individual amide protons. The rate constant distributions determined by both methods are consistent and complementary, thereby validating protein fragmentation/mass spectrometry as a reliable measure of hydrogen exchange in proteins.
Protein—protein crystal‐packing contacts
Authors: Carugo Oliviero , Argos Patrick 
Published: February 2009

AbstractProtein‐protein contacts in monomeric protein crystal structures have been analyzed and compared to the physiological protein‐protein contacts in oligomerization. A number of features differentiate the crystal‐packing contacts from the natural contacts occurring in multimeric proteins. The area of the protein surface patches involved in packing contacts is generally smaller and its amino acid composition is indistinguishable from that of the protein surface accessible to the solvent. The fraction of protein surface in crystal contacts is very variable and independent of the number of packing contacts. The thermal motion at the crystal packing interface is intermediate between that of the solvent‐accessible surface and that of the protein core, even for large packing interfaces, though the tendency is to be closer to that of the core. These results suggest that protein crystallization depends on random protein‐protein interactions, which have little in common with physiological protein‐protein recognition processes, and that the possibility of engineering macromolecular crystallization to improve crystal quality could be widened.
Identification, expression, and crystallization of the protease‐resistant conserved domain of synapsin I
Authors: Wang Chyung‐Ru , Esser Lothar , Smagula Cynthia S. , Südhof Thomas C. , Deisenhofer Johann 
Published: February 2009

AbstractA 35‐37‐kDa protease‐resistant domain of synapsin Ia/Ib, apparently produced by low levels of endogenous proteases in vapor diffusion droplets, slowly formed crystals diffracting X‐rays to ∼ 10 Å resolution. The fragment mainly consisted of the highly conserved C domain common to the synapsin I/II family plus short N‐ and C‐terminal flanking segments. Two constructs (SynA and SynB) of synthetic gene fragments coding for the C domain of synapsin with or without C‐terminal flanking sequence were expressed in Escherichia coli as fusion proteins attached to the soluble protein glutathione‐S‐transferase. The fusion proteins were purified by affinity chromatography. Subsequent in situ cleavage with TEV protease resulted in the release of highly pure synapsin fragments, which were further purified by ion exchange chromatography. SynA and SynB formed crystals within three days, which diffracted to better than 3 Å using a conventional X‐ray source and to about 2 Å using a synchrotron X‐ray source. SynA crystals have the symmetry of the trigonal space groups P3121 or P3221 and the unit cell dimensions a = b = 77.4 Å, c = 188.5 Å, α = β = 90°, γ = 120°. SynB crystals have the symmetry of the orthorhombic space group C2221 with the unit cell dimensions a = 104.6 Å, b = 113.3 Å, and c = 273.8 Å.
Crystal structure of cyclophilin A complexed with a binding site peptide from the HIV‐1 capsid protein
Authors: Vajdos Felix F. , Yoo Sanghee , Houseweart Megan , Sundquist Wesley I. , Hill Christopher P. 
Published: February 2009

AbstractThe cellular protein, cyclophilin A (CypA), is incorporated into the virion of the type 1 human immunodeficiency virus (HIV‐1) via a direct interaction with the capsid domain of the viral Gag polyprotein. We demonstrate that the capsid sequence 87His‐Ala‐Gly‐Pro‐Ile‐Ala92 (87HAGPIA92) encompasses the primary cyclophilin A binding site and present an X‐ray crystal structure of the CypA/HAGPIA complex. In contrast to the cis prolines observed in all previously reported structures of CypA complexed with model peptides, the proline in this peptide, Pro 90, binds the cyclophilin A active site in a trans conformation. We also report the crystal structure of a complex between CypA and the hexapeptide HVGPIA, which also maintains the trans conformation. Comparison with the recently determined structures of CypA in complexes with larger fragments of the HIV‐1 capsid protein demonstrates that CypA recognition of these hexapeptides involves contacts with peptide residues Ala(Val) 88, Gly 89, and Pro 90, and is independent of the context of longer sequences.
Tess: A geometric hashing algorithm for deriving 3D coordinate templates for searching structural databases. Application to enzyme active sites
Authors: Wallace Andrew C. , Borkakoti Neera , Thornton Janet M. 
Published: February 2009

AbstractIt is well established that sequence templates such as those in the PROSITE and PRINTS databases are powerful tools for predicting the biological function and tertiary structure for newly derived protein sequences. The number of X‐ray and NMR protein structures is increasing rapidly and it is apparent that a 3D equivalent of the sequence templates is needed. Here, we describe an algorithm called TESS that automatically derives 3D templates from structures deposited in the Brookhaven Protein Data Bank. While a new sequence can be searched for sequence patterns, a new structure can be scanned against these 3D templates to identify functional sites. As examples, 3D templates are derived for enzymes with an O‐His‐O “catalytic triad” and for the ribonucleases and lysozymes. When these 3D templates are applied to a large data set of nonidentical proteins, several interesting hits are located. This suggests that the development of a 3D template database may help to identify the function of new protein structures, if unknown, as well as to design proteins with specific functions.
Kinase conformations: A computational study of the effect of ligand binding
Authors: Helms Volkhard , McCammon J. Andrew 
Published: February 2009

AbstractProtein function is often controlled by ligand‐induced conformational transitions. Yet, in spite of the increasing number of three‐dimensional crystal structures of proteins in different conformations, not much is known about the driving forces of these transitions. As an initial step toward exploring the conformational and energetic landscape of protein kinases by computational methods, intramolecular energies and hydration free energies were calculated for different conformations of the catalytic domain of CAMP‐dependent protein kinase (cAPK) with a continuum (Poisson) model for the electrostatics. Three protein kinase crystal structures for ternary complexes of cAPK with the peptide inhibitor PKI(5‐24) and ATP or AMP‐PNP were modeled into idealized intermediate and open conformations. Concordant with experimental observation, we find that the binding of PKI(5‐24) is more effective in stabilizing the closed and intermediate forms of cAPK than ATP. PKI(5‐24) seems to drive the final closure of the active site cleft from intermediate to closed state because ATP does not distinguish between these two states. Binding of PKI(5‐24) and ATP is energetically additive.
Green fluorescent protein as a signal for protein–protein interactions
Authors: Park Sang‐Hyun , Raines Ronald T. 
Published: February 2009

AbstractGreen fluorescent protein (GFP) is autofluorescent. This property has made GFP useful in monitoring in vivo activities such as gene expression and protein localization. We find that GFP can be used in vitro to reveal and characterize protein‐protein interactions. The interaction between the S‐peptide and S‐protein fragments of ribonuclease A was chosen as a model system. GFP‐tagged S‐peptide was produced, and the interaction of this fusion protein with S‐protein was analyzed by two distinct methods: fluorescence gel retardation and fluorescence polarization. The fluorescence gel retardation assay is a rapid method to demonstrate the existence of a protein‐protein interaction and to estimate the dissociation constant (Kd) of the resulting complex. The fluorescence polarization assay is an accurate method to evaluate Kd in a specified homogeneous solution and can be adapted for the high‐throughput screening of protein or peptide libraries. These two methods are powerful new tools to probe protein‐protein interactions.
Molecular mechanics calculations on rous sarcoma virus protease with peptide substrates
Authors: Weber Irene T. , Harrison Robert W. 
Published: February 2009

AbstractMolecular models of Rous sarcoma virus (RSV) protease and 20 peptide substrates with single amino acid substitutions at positions from P4 to P3′, where the scissile bond is between P1 and Pl′, were built and compared with kinetic measurements. The unsubstituted peptide substrate, Pro‐Ala‐Val‐Ser‐Leu‐Ala‐Met‐Thr, represents the NC‐PR cleavage site of RSV protease. Models were built of two intermediates in the catalytic reaction, RSV protease with peptide substrate and with the tetrahedral intermediate. The energy minimization used an algorithm that increased the speed and eliminated a cutoff for nonbonded interactions. The calculated protease‐substrate interaction energies showed correlation with the relative catalytic efficiency of peptide hydrolysis. The calculated interaction energies for the 8 RSV protease‐substrate models with changes in P1 to P1′ next to the scissile bond gave the highest correlation coefficient of 0.79 with the kinetic measurements, whereas all 20 substrates showed the lower, but still significant correlation of 0.46. Models of the tetrahedral reaction intermediates gave a correlation of 0.72 for the 8 substrates with changes next to the scissile bond, whereas a correlation coefficient of only 0.34 was observed for all 20 substrates. The differences between the energies calculated for the tetrahedral intermediate and the bound peptide gave the most significant correlation coefficients of 0.90 for models with changes in P1 and P1′, and 0.56 for all substrates. These results are compared to those from similar calculations on HIV‐1 protease and discussed in relation to the rate‐limiting steps in the catalytic mechanism and the entropic contributions.