bluuebunny/crossref_metadata_2025 huggingface dataset:
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

Thymidine 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

The 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

Clusterin 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

Temperature‐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

Glutathione 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

The 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

Hydrogen/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

Protein‐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

A 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

The 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

It 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

Protein 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

Green 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

Molecular 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.
1H, 13C, and 15N resonance assignments of Fusarium solani pisi cutinase and preliminary features of the structure in solution
Authors: Prompers Jeanine J. , Hilbers Cornelis W. , Groenewegen Anneke , Schaik René C. Van , Pepermans Henri A. M.
Published: February 2009

Essentially complete (96%) sequence‐specific assignments were made for the backbone and side‐chain 1H, 13C, and 15N resonances of Fusarium solani pisi cutinase, produced as a 214‐residue heterologous protein in Escherichia coli, using heteronuclear NMR techniques. Three structural features were noticed during the assignment. (1) The secondary structure in solution corresponds mostly with the structure from X‐ray diffraction, suggesting that both structures are globally similar. (2) The HN of Ala32 has a strongly upfield‐shifted resonance at 3.97 ppm, indicative of an amide‐aromatic hydrogen bond to the indole ring of Trp69 that stabilizes the N‐terminal side of the parallel β‐sheet. (3) The NMR data suggest that the residues constituting the oxyanion hole are quite mobile in the free enzyme in solution, in contrast to the existence of a preformed oxyanion hole as observed in the crystal structure. Apparently, cutinase forms its oxyanion hole upon binding of the substrate like true lipases.
Domain organization of calbindin D28k as determined from the association of six synthetic EF‐hand fragments
Authors: Linse Sara , Thulin Eva , Gifford Lida K. , Radzewsky Dina , Hagan James , Åkerfeldt Karin S. , Wilk Rosemarie R.
Published: February 2009

Calbindin D28k is an intracellular Ca2+‐binding protein containing six subdomains of EF‐hand type. The number and identity of the globular domains within this protein have been elucidated using six synthetic peptide fragments, each corresponding to one EF‐hand subdomain. All six peptides were mixed in equimolar amounts in the presence of 10 mM Ca2+ to allow for the reconstitution of domains. The mixture was compared to native calbindin D28k and to the sum of the properties of the individual peptides using circular dichroism (CD), fluorescence, and 1H NMR spectroscopy, as well as gel filtration and ion‐exchange chromatography. It was anticipated that if the peptides associate to form native‐like domains, the properties would be similar to those of the intact protein, whereas if they did not interact, they would be the same as the properties of the isolated peptides. The results show that the peptides in the mixture interact with one another. For example, the CD and fluorescence spectra for the mixture are very similar to those of the intact calbindin D28k suggesting that the mixed EF‐hand fragments associate to form a native‐like structure. To determine the number of domains and the subdomain composition of each domain in calbindin D28k, a variety of peptide combinations containing two to five EF‐hand fragments were studied. The spectral and chromatographic properties of all the mixtures containing less than six peptides were closer to the sum of the properties of the relevant individual peptides than to the mixture of the six peptides. The results strongly suggest that all six EF‐hands are packed into one globular domain. The association of the peptide fragments is observed to drive the folding of the individual subdomains. For example, one of the fragments, EF2, which is largely unstructured in isolation even in the presence of high concentrations of Ca2+, is considerably more structured in the presence of the other peptides, as judged by CD difference spectroscopy. The CD data also suggest that the packing between the individual subdomains is specific.
Covalent methionylation of escherichia coli methionyl‐tRNA synthethase: Identification of the labeled amino acid residues by matrix‐assisted laser desorption‐ionization mass spectrometry
Authors: Gillet Sylvie , Hountondji Codjo , Schmitter Jean‐Marie , Blanquet Sylvain
Published: February 2009

Methionyl‐adenylate, the mixed carboxylic‐phosphoric acid anhydride synthesized by methionyl‐tRNA synthetase (MetRS) is capable of reacting with this synthetase or other proteins, by forming an isopeptide bond with the ϵ‐NH2 group of lysyl residues. It is proposed that the mechanism for the in vitro methionylation of MetRS might be accounted for by the in situ covalent reaction of methionyl‐adenylate with lysine side chains surrounding the active center of the enzyme, as well as by exchange of the label between donor and acceptor proteins. Following the incorporation of 7.0 ± 0.5 mol of methionine per mol of a monomeric truncated methionyl‐tRNA synthetase species, the enzymic activities of [32P]PPi‐ATP isotopic exchange and tRNAMet aminoacylation were lowered by 75% and more than 90%, respectively. The addition of tRNAMet protected the enzyme against inactivation and methionine incorporation. Matrix‐assisted laser desorption‐ionization mass spectrometry designated lysines‐114, ‐132, ‐142 (or ‐147), ‐270, ‐282, ‐335, ‐362, ‐402, ‐439, ‐465, and ‐547 of truncated methionyl‐tRNA synthetase as the target residues for covalent binding of methionine. These lysyl residues are distributed at the surface of the enzyme between three regions [114‐150], [270‐362], and [402‐465], all of which were previously shown to be involved in catalysis or to be located in the binding sites of the three substrates, methionine, ATP, and tRNA.
Direct sequence analysis of proteins by in‐source fragmentation during delayed ion extraction
Authors: Lennon John J. , Walsh Kenneth A.
Published: February 2009

Continuous segments of amino acid sequence information as long as 41 residues have been deduced by interpretation of matrix‐assisted laser desorption/ionization‐generated ion signals dominated by cn fragmentation within the ion source of a linear time‐of‐flight mass spectrometer utilizing delayed ion extraction. The technique has been applied successively to five proteins of mass 12.2 kDa to 18.3 kDa, yielding segments of continuous sequence as long as 41 residues without the need for prior proteolytic fragmentation. Intact crosslinks such as disulfides or heme linkages interrupt the generation of these data.
Identification of a novel conserved sequence motif in flavoprotein hydroxylases with a putative dual function in FAD/NAD(P)H binding
Authors: Eppink Michel H. M. , Berkel Willem J. H. Van , Schreuder Herman A.
Published: February 2009

A novel conserved sequence motif has been located among the flavoprotein hydroxylases. Based on the crystal structure and site‐directed mutagenesis studies of p‐hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescens, this amino acid fingerprint sequence is proposed to play a dual function in both FAD and NAD(P)H binding. In PHBH, the novel sequence motif (residues 153‐166) includes strand A4 and the N‐terminal part of helix H7. The conserved amino acids Asp 159, Gly 160, and Arg 166 are necessary for maintaining the structure. The backbone oxygen of Cys 158 and backbone nitrogens of Gly 160 and Phe 161 interact indirectly with the pyrophosphate moiety of FAD, whereas it is known from mutagenesis studies that the side chain of the moderately conserved His 162 is involved in NADPH binding.
Preliminary crystal structure determination of the alkaline protease from the Antarctic psychrophile pseudomonas aeruginosa
Authors: Villeret Vincent , Beeumen Jozef Van , Chessa Jean‐Pierre , Gerday Charles
Published: February 2009

A cold alkaline protease, isolated from an Antarctic Pseudomonas aeruginosa strain, has been purified and crystallized. Large crystals were obtained in the presence of PEG 6000 at pH 7 and pH 8. They belong to the space group P212121. A complete data set to 2.1 Å resolution has been measured. The structure has been determined by the molecular replacement method using the coordinates of the mesophilic alkaline protease as a model. The molecular replacement solution displays a correlation coefficient of 0.39 and an R‐factor of 0.48. Subsequent inspection of the electron density map in the active site region has confirmed the correctness of the solution. Model building and structure refinement will be initiated when the protease sequence becomes fully available. This is the second report, following one on an a‐amylase, of the preliminary crystallographic characterization of a psychrophilic enzyme.
Purification, characterization, and crystallization of Escherichia coli ribokinase
Authors: Sigrell Jill A. , Cameron Alexander D. , Jones T. Alwyn , Mowbray Sherry L.
Published: February 2009

Ribokinase phosphorylates ribose to form ribose‐5‐phosphate in the presence of ATP and magnesium. The phosphorylated sugar can enter the pentose phosphate pathway or be used for the synthesis of nucleotides, histidine, and tryptophan. Ribokinase belongs to the PfkB family of carbohydrate kinases, for which no three‐dimensional structure is currently known. We describe an improved purification protocol for Escherichia coli ribokinase and give evidence from light‐scattering and gel filtration studies that the protein forms a dimer in solution. Several types of crystals are also described that have been obtained of apo ribokinase, ribokinase in the presence of ATP, and in a ternary complex with an ATP‐analogue and ribose. The latter crystals give the best X‐ray diffraction. A complete data set has been collected at the synchrotron source in Hamburg, to 2.6 Å resolution using a frozen crystal. The crystals belong to space group P6122 or P6522 with cell parameters a = b = 95 Å and c = 155 Å.
The partial charge of the nitrogen atom in peptide bonds
Authors: Milner‐White E. James
Published: February 2009

A majority of the standard texts dealing with proteins portray the peptide link as a mixture of two resonance forms, in one of which the nitrogen atom has a positive charge. As a consequence, it is often believed that the nitrogen atom has a net positive charge. This is in apparent contradiction with the partial negative charge on the nitrogen that is used in force fields for molecular modeling. However, charges on resonance forms are best regarded as formal rather than actual charges and current evidence clearly favors a net negative charge for the nitrogen atom. In the course of the discussion, new ideas about the electronic structure of amides and the peptide bond are presented.
Identification of the binding site on S100B protein for the actin capping protein CapZ
Authors: Kilby Peter M. , Roberts GORDON C. K. , Van Eldik Linda J.
Published: February 2009

The calcium‐binding protein S100B binds to several potential target proteins, but there is no detailed information showing the location of the binding site for any target protein on S100B. We have made backbone assignments of the calcium‐bound form of S100B and used chemical‐shift changes in spectra of 15N‐labeled protein to locate the site that binds a peptide corresponding to residues 265‐276 from CapZα, the actin capping protein. The largest chemical‐shift changes are observed for resonances arising from residues around the C terminus of the C‐terminal helix of S100B and residues Val‐8 to Asp‐12 of the N‐terminal helix. These residues are close to but not identical to residues that have been identified by mutational analysis to be important in other S100 protein‐protein interactions. They make up a patch across the S100B dimer interface and include some residues that are quite buried in the structure of calcium‐free S100B. We believe we may have identified a binding site that could be common to many S100 protein—protein interactions.
De novo heme proteins from designed combinatorial libraries
Authors: Hecht Michael H. , Vogel Kathleen M. , Spiro Thomas G. , Rojas Nina R. L. , Kamtekar Satwik , Simons Cyrena T. , Mclean Jeremy E. , Farid Ramy S.
Published: February 2009

We previously reported the design of a library of de novo amino acid sequences targeted to fold into four‐helix bundles. The design of these sequences was based on a “binary code” strategy, in which the patterning of polar and non‐polar amino acids is specified explicitly, but the exact identities of the side chains is varied extensively (Kamtekar S, Schiffer JM, Xiong H, Babik JM, Hecht MH, 1993, Science 262:1680‐1685). Because of this variability, the resulting collection of amino acid sequences may include de novo proteins capable of binding biologically important cofactors. To probe for such binding, the de novo sequences were screened for their ability to bind the heme cofactor. Among an initial collection of 30 binary code sequences, 15 are shown to bind heme and form bright red complexes. Characterization of several of these de novo heme proteins demonstrated that their absorption spectra and resonance Raman spectra resemble those of natural cytochromes. Because the design of these sequences is based on global features of polar/nonpolar patterning, the finding that half of them bind heme highlights the power of the binary code strategy, and demonstrates that isolating de novo heme proteins does not require explicit design of the cofactor binding site. Because bound heme plays a key role in the functions of many natural proteins, these results suggest that binary code sequences may serve as initial prototypes for the development of large collections of functionally active de novo proteins.
Linkers of secondary structures in proteins
Authors: Geetha V. , Munson P. J.
Published: February 2009

Linkers that connect repeating secondary structures fall into conformational classes based on distance and main‐chain torsion clustering. A data set of 300 unique protein chains with low pairwise sequence identity was clustered into only a few groups representing the preferred motifs. The linkers of two to eight residues for the nonredundant data set are designated H‐Ln‐H, H‐Ln‐E, E‐Ln‐H, E‐Ln‐E, where n is the length, H stands for α‐helices, and E for β‐strands. Most of the clusters identified here corroborate earlier findings. However, 19 new clusters are identified in this paper, with many of them having seven and eight residue linkers. In our first analysis, the secondary structures flanking the linkers are both interacting and noninteracting and there is no precise angle of orientation between them. A second analysis was performed on a set of proteins with restricted orientations for the flanking elements, namely, mainly α class of proteins with orthogonal architecture. Two definite clusters are identified, one corresponding to linkers of orthogonal helices and the other to linkers of antiparallel helices. Loops forming binding sites or involved in catalytic activity are important determinants of the function of proteins. Although the structural conservation of the residues around the catalytic triad of serine proteases has been studied widely, there has not been a systematic analysis of the conformation of the loops that contain them. Residues of the catalytic triad reside in the linkers of β‐strands, with varying lengths of more than eight residues. Here, we analyze the structural conservation of such linkers by superposition, and observe a conserved structural feature of the linkers incorporating each of the three residues of the catalytic triad.
Cross‐strand side‐chain interactions versus turn conformation in β‐hairpins
Authors: Alba Eva De , Rico Manuel , Jiménez M. Angeles
Published: February 2009

A series of designed peptides has been analyzed by 1H‐NMR spectroscopy in order to investigate the influence of cross‐strand side‐chain interactions in β‐hairpin formation. The peptides differ in the N‐terminal residues of a previously designed linear decapeptide that folds in aqueous solution into two interconverting β‐hairpin conformations, one with a type I turn (β‐hairpin 4:4) and the other with a type I + G1 β‐bulge turn (β‐hairpin 3:5). Analysis of the conformational behavior of the peptides studied here demonstrates three favorable and two unfavorable cross‐strand side‐chain interactions for β‐hairpin formation. These results are in agreement with statistical data on side‐chain interactions in protein β‐sheets. All the peptides in this study form significant populations of the β‐hairpin 3:5, but only some of them also adopt the β‐hairpin 4:4. The formation of β‐hairpin 4:4 requires the presence of at least two favorable cross‐strand interactions, whereas β‐hairpin 3:5 seems to be less susceptible to side‐chain interactions. A protein database analysis of β‐hairpins 3:5 and β‐hairpins 4:4 indicates that the former occur more frequently than the latter. In both peptides and proteins, β‐hairpins 3:5 have a larger right‐handed twist than β‐hairpins 4:4, so that a factor contributing to the higher stability of β‐hairpin 3:5 relative to β‐hairpin 4:4 is due to an appropriate backbone conformation of the type I + G1 β‐bulge turn toward the right‐handed twist usually observed in protein β‐sheets. In contrast, as suggested previously, backbone geometry of the type I turn is not adequate for the right‐handed twist. Because analysis of buried hydrophobic surface areas on protein β‐hairpins reveals that β‐hairpins 3:5 bury more hydrophobic surface area than β‐hairpins 4:4, we suggest that the right‐handed twist observed in β‐hairpin 3:5 allows a better packing of side chains and that this may also contribute to its higher intrinsic stability.
Analysis of temperature factor distribution in high‐resolution protein structures
Authors: Parthasarathy S. , Murthy M.R.N.
Published: February 2009

The temperature factors obtained from X‐ray refinement of proteins at high resolution show large variations from one structure to another. However, the B‐values expressed in units of standard deviation about their mean value (B'‐factor) at the Cα atoms show remarkably characteristic frequency distribution. In all of the 110 proteins examined in this study, the frequency distribution exhibited a bimodal distribution. The peaks in the B'‐factor frequency distribution occur at ‐1.1 and 0.4 for a bin size of 0.5. The peak at lower temperature factor corresponds largely to buried residues, whereas the peak at larger value corresponds to exposed residues. The distribution could be accurately described as a superposition of two Gaussian functions. The parameters describing the distribution are therefore characteristic of protein structures. The frequency distribution for a given amino acid over all the proteins also shows a similar bimodal distribution, although the areas under the two Gaussians differ from one amino acid to another. The area under the frequency distribution curve for any interval in B'‐factor represents the propensity of the amino acid to occur in that interval. This propensity is related both to the hydrophilicity/hydrophobicity of the residue and the tendency of the residue to impose a different degree of rigidity on the polypeptide chain. The frequency distribution of stretches of high B'‐factors departs appreciably from that expected for a random distribution. The correlation in the B‐values of sequentially proximal residues is probably responsible for the bimodal distribution.
Simulating the minimum core for hydrophobic collapse in globular proteins
Authors: Tsai Jerry , Gerstein Mark , Levitt Michael
Published: February 2009

To investigate the nature of hydrophobic collapse considered to be the driving force in protein folding, we have simulated aqueous solutions of two model hydrophobic solutes, methane and isobutylene. Using a novel methodology for determining contacts, we can precisely follow hydrophobic aggregation as it proceeds through three stages: dispersed, transition, and collapsed. Theoretical modeling of the cluster formation observed by simulation indicates that this aggregation is cooperative and that the simulations favor the formation of a single cluster midway through the transition stage. This defines a minimum solute hydrophobic core volume. We compare this with protein hydrophobic core volumes determined from solved crystal structures. Our analysis shows that the solute core volume roughly estimates the minimum core size required for independent hydrophobic stabilization of a protein and defines a limiting concentration of nonpolar residues that can cause hydrophobic collapse. These results suggest that the physical forces driving aggregation of hydrophobic molecules in water is indeed responsible for protein folding.
Thermal unfolding of dodecameric glutamine synthetase: Inhibition of aggregation by urea
Authors: Nosworthy Neil J. , Ginsburg Ann
Published: February 2009

Thermal unfolding of dodecameric manganese glutamine synthetase (622,000 Mr) at pH 7 and ∼0.02 ionic strength occurs in two observable steps: a small reversible transition (Tm ∼ 42°C; ΔH ≅ 0.9 J/g) followed by a large irreversible transition (Tm ∼ 81°C; ΔH≅ 23.4 J/g) in which secondary structure is lost and soluble aggregates form. Secondary structure, hydrophobicity, and oligomeric structure of the equilibrium intermediate are the same as for the native protein, whereas some aromatic residues are more exposed. Urea (3 M) destabilizes the dodecamer (with a tertiary structure similar to that without urea at 55°C) and inhibits aggregation accompanying unfolding at ≤50.2 mg protein/mL. With increasing temperature (30‐70°C) or incubation times at 25°C (5‐35 h) in 3 M urea, only dodecamer and unfolded monomer are detected. In addition, the loss in enzyme secondary structure is pseudo‐first‐order (t1/2 = 1,030s at 20.0°C in 4.5 M urea). Differential scanning calorimetry of the enzyme in 3 M urea shows one endotherm (Tmax ∼ 64°C; ΔH = 17 ± 2 J/g). The enthalpy change for dissociation and unfolding agrees with that determined by urea titrations by isothermal calorimetry (ΔH = 57 ± 15 J/g; Zolkiewski M, Nosworthy NJ, Ginsburg A, 1995, Protein Sci 4:144‐1552), after correcting for the binding of urea to protein sites exposed during unfolding (‐42 J/g). Refolding and assembly to active enzyme occurs upon dilution of urea after thermal unfolding.
Demonstration of protein—protein interaction specificity by NMR chemical shift mapping
Authors: Rajagopal Ponni , Waygood E. Bruce , Reizer Jonathan , Saier Milton H. , Klevit Rachel E.
Published: February 2009

Chemical shift mapping is becoming a popular method for studying protein‐protein interactions in solution. The technique is used to identify putative sites of interaction on a protein surface by detecting chemical shift perturbations in simple (1H, 15N )‐HSQC NMR spectra of a uniformly labeled protein as a function of added (unlabeled) target protein. The high concentrations required for these experiments raise questions concerning the possibility for non‐specific interactions being detected, thereby compromising the information obtained. We demonstrate here that the simple chemical shift mapping approach faithfully reproduces the known functional specificities among pairs of closely related proteins from the phosphoenolpyruvate:sugar phosphotransferase systems of Escherichia coli and Bacillus subtilis.
OLDERADO: On‐line database of ensemble representatives and domains
Authors: Kelley Lawrence A. , Sutcliffe Michael J.
Published: February 2009

In cases where the structure of a single protein is represented by an ensemble of conformations, there is often a need to determine the common features and to choose a “representative” conformation. This occurs, for example, with structures determined by NMR spectroscopy, analysis of the trajectory from a molecular dynamics simulation, or an ensemble of structures produced by comparative modeling. We reported previously automatic methods for (1) defining the atoms with low spatial variance across an ensemble (i.e., the “core” atoms) and the domains in which these atoms lie, and (2) clustering an ensemble into conformationally related subfamilies. To extend the utility of these methods, we have developed a freely available server on the World Wide Web at http://neon.chem.le.ac.uk/olderado/. This (1) contains an automatically generated database of representative structures, core atoms, and domains determined for 449 ensembles of NMR‐derived protein structures in the Protein Data Bank (PDB) in May 1997, and (2) allows the user to upload a PDB‐formatted file containing the coordinates of an ensemble of structures. The server returns in real time: (1) information on the residues constituting domains; (2) the structures that constitute each conformational subfamily; and (3) an interactive java‐based three‐dimensional viewer to visualise the domains and clusters. Such information is useful, for example, when selecting conformations to be used in comparative modeling and when choosing parts of structures to be used in molecular replacement. Here we describe the OLDERADO server.
A diverse superfamily of enzymes with ATP‐dependent carboxylate—amine/thiol ligase activity
Authors: Galperin Michael Y. , Koonin Eugene V.
Published: February 2009

The recently developed PSI‐BLAST method for sequence database search and methods for motif analysis were used to define and expand a superfamily of enzymes with an unusual nucleotide‐binding fold, referred to as palmate, or ATP‐grasp fold. In addition to d‐alanine‐d‐alanine ligase, glutathione synthetase, biotin carboxylase, and carbamoyl phosphate synthetase, enzymes with known three‐dimensional structures, the ATP‐grasp domain is predicted in the ribosomal protein S6 modification enzyme (RimK), urea amidolyase, tubulin‐tyrosine ligase, and three enzymes of purine biosynthesis. All these enzymes possess ATP‐dependent carboxylate‐amine ligase activity, and their catalytic mechanisms are likely to include acylphosphate intermediates. The ATP‐grasp superfamily also includes succinate‐CoA ligase (both ADP‐forming and GDP‐forming variants), malate‐CoA ligase, and ATP‐citrate lyase, enzymes with a carboxylate‐thiol ligase activity, and several uncharacterized proteins. These findings significantly extend the variety of the substrates of ATP‐grasp enzymes and the range of biochemical pathways in which they are involved, and demonstrate the complementarity between structural comparison and powerful methods for sequence analysis.