bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparison of straight chain and cyclic unnatural amino acids embedded in the core of staphylococcal nuclease
Authors: Richards Frederic M. , Wynn Richard , Harkins Paul C. , Fox Robert O. 
Published: February 2009

AbstractWe have determined by X‐ray crystallography the structures of several variants of staphylococcal nuclease with long flexible straight chain and equivalent length cyclic unnatural amino acid side chains embedded in the protein core. The terminal atoms in the straight side chains are not well defined by the observed electron density even though they remain buried within the protein interior. We have previously observed this behavior and have suggested that it may arise from the addition of side‐chain vibrational and oscillational motions with each bond as a side chain grows away from the relatively rigid protein main chain and/or the population of multiple rotamers (Wynn R, Harkins P, Richards FM, Fox RO. 1996. Mobile unnatural amino acid side chains in the core of staphylococcal nuclease. Protein Sci 5:1026‐1031). Reduction of the number of degrees of freedom by cyclization of a side chain would be expected to constrain these motions. These side chains are in fact well defined in the structures described here. Over‐packing of the protein core results in a 1.O Å shift of helix 1 away from the site of mutation. Additionally, we have determined the structure of a side chain containing a single hydrogen to fluorine atom replacement on a methyl group. A fluorine atom is intermediate in size between methyl group and a hydrogen atom. The fluorine atom is observed in a single position indicating it does not rotate like methyl hydrogen atoms. This change also causes subtle differences in the packing interactions.
Bayesian statistical analysis of protein side‐chain rotamer preferences
Authors: Dunbrack Roland L. , Cohen Fred E. 
Published: February 2009

AbstractWe present a Bayesian statistical analysis of the conformations of side chains in proteins from the Protein Data Bank. This is an extension of the backbone‐dependent rotamer library, and includes rotamer populations and average x angles for a full range of ø,ψ values. The Bayesian analysis used here provides a rigorous statistical method for taking account of varying amounts of data. Bayesian statistics requires the assumption of a prior distribution for parameters over their range of possible values. This prior distribution can be derived from previous data or from pooling some of the present data. The prior distribution is combined with the data to form the posterior distribution, which is a compromise between the prior distribution and the data. For the X2, X3, and X4 rotamer prior distributions, we assume that the probability of each rotamer type is dependent only on the previous X rotamer in the chain. For the backbone‐dependence of the x1 rotamers, we derive prior distributions from the product of the ø‐dependent and ψ‐dependent probabilities. Molecular mechanics calculations with the CHARMM22 potential show a strong similarity with the experimental distributions, indicating that proteins attain their lowest energy rotamers with respect to local backbone‐side‐chain interactions. The new library is suitable for use in homology modeling, protein folding simulations, and the refinement of X‐ray and NMR structures.
The role of PII conformations in the calculation of peptide fractional helix content
Authors: Park Soon‐Ho , Shalongo William , Stellwagen Earle 
Published: February 2009

AbstractChanges in the temperature, pH, ionic strength, or denaturant concentration of aqueous solutions of the monomeric non‐α‐helical peptide acety1YEAAAKEAPAKEAAAKAamide generate changes in its dichroic spectrum characteristic for a conformational transition. This transition has the characteristic features of a residue PII/unstructured conformational equilibrium in which PII denotes an extended left‐handed helical conformation and unstructured denotes all the remaining conformations in a random coil ensemble. Replacement of the proline residue facilitates population of residues in an α‐helical conformation. However, the elliptcity values for these non‐proline peptides merge with the ellipticity of the proline peptide as the population of residues in the α‐helix conformation is diminished. This convergence suggests that all residues in a host/guest peptide series of the same length share a common PII/unstructured conformational equilibrium in a given solvent. We propose that the fractional helix content of peptides within such a series may be estimated by using a two‐state calculation in which the ellipticity for the non‐α‐helix conformations is provided by a peptide having a central proline guest residue.
Xylanase XynA from the hyperthermophilic bacterium Thermotoga maritima: Structure and stability of the recombinant enzyme and its isolated cellulose‐binding domain
Authors: Wassenberg Doris , Schurig Hartmut , Jaenicke Rainer , Liebl Wolfgang 
Published: February 2009

AbstractThe hyperthermophilic bacterium Thermotoga maritima is capable of gaining metabolic energy utilizing xylan. XynA, one of the corresponding hydrolases required for its degradation, is a 120‐kDa endo‐l,4‐D‐xylanase exhibiting high intrinsic stability and a temperature optimum —90 °C. Sequence alignments with other xylanases suggest the enzyme to consist of five domains. The C‐terminal part of XynA was previously shown to be responsible for cellulose binding (Winterhalter C, Heinrich P, Candussio A, Wich G, Liebl W. 1995. Identification of a novel cellulose‐binding domain within the multi‐domain 120 kDa Xylanase XynA of the hyperthermophilic bacterium Thermotoga maritima. Mol Microbiol 15:431‐444).In order to characterize the domain organization and the stability of XynA and its C‐terminal cellulose‐binding domain (CBD), the two separate proteins were expressed in Escherichia coli. CBD, because of its instability in its ligand‐free form, was expressed as a glutathione S‐transferase fusion protein with a specific thrombin cleavage site as linker. XynA and CBD were compared regarding their hydrodynamic and spectral properties. As taken from analytical ultracentrifugation and gel permeation chromatography, both are monomers with 116 and 22 kDa molecular masses, respectively. In the presence of glucose as a ligand, CBD shows high intrinsic stability. Denaturation/renaturation experiments with isolated CBD yield >80% renaturation, indicating that the domain folds independently. Making use of fluorescence emission and far‐UV circular dichroism in order to characterize protein stability, guanidine‐induced unfolding of XynA leads to biphasic transitions, with half‐concentrations c1/2(GdmC1) ∼4 M and >5 M, in accordance with the extreme thermal stability. At acid pH, XynA exhibits increased stability, indicated by a shift of the second guanidine‐transition from 5 to 7 M GdmC1. This can be tentatively attributed to the cellulose‐binding domain. Differences in the transition profiles monitored by fluorescence emission and dichroic absorption indicate multi‐state behavior of XynA. In the case of CBD, a temperature‐induced increase in negative ellipticity at 217 nm is caused by alterations in the environment of aromatic residues that contribute to the far‐UV CD in the native state.
An unexpected structural relationship between integral membrane phosphatases and soluble haloperoxidases
Authors: Neuwald Andrew F. 
Published: February 2009

AbstractThe mechanism of a membrane‐bound enzyme important in phospholipid signaling, type 2 phosphatidic acid phosphatase, is suggested by sequence motifs shared with a soluble vanadium‐dependent chloroperoxidase of known structure. These regions are also conserved in other soluble globular and membrane‐associated proteins, including bacterial acid phosphatases, mammalian glucose‐6‐phosphatases, and the Drosophila developmental protein Wunen. This implies that a similar arrangement of catalytic residues specifies the active site within both soluble and membrane spanning domains.
CD6 recognizes the neural adhesion molecule BEN
Authors: Skonier John E. , Bowen Michael A. , Aruffo Alejandro , Bajorath JÜRgen 
Published: February 2009

AbstractCD6 and its ligand activated leukocyte cell adhesion molecule (ALCAM, CD166) have been detected on various immune cells and in the brain. CD6‐ligand interactions have been implicated in the regulation of T cell function. ALCAM shares the same extracellular domain organization and significant sequence homology with the chicken neural adhesion molecule BEN. Although ALCAM's CD6 binding site is only partially conserved in BEN, CD6 specifically binds BEN, albeit with ˜ 10‐fold lower avidity than ALCAM. Differences in binding avidity are not detected when ALCAM and BEN fusion proteins containing the full‐length extracellular regions are tested. Homotypic interactions between full‐length forms are likely to account for these observations. The identified cross‐species interaction between CD6 and BEN suggests that CD6‐ligand interactions are highly conserved.
Crystallization of 5‐aminolaevulinic acid dehydratase from Escherichia coli and Saccharomyces cerevisiae and preliminary X‐ray characterization of the crystals
Authors: Erskine P.T. , Cooper J. , Lambert R. , Lewis G. , Wood S.P. , Shoolingin‐Jordan P.M. , Maignan S. , Spencer P. , Senior N. , Awan S. , Warren M. , Tickle I.J. , Thomas P. 
Published: February 2009

Abstract5‐Aminolaevulinic acid dehydratase (ALAD) catalyzes the formation of porphobilinogen from two molecules of 5‐aminolaevulinic acid. Both Escherichia coli and Saccharomyces cerevisiae ALADs are homo‐octameric enzymes which depend on Zn2+ for catalytic activity and are potently inhibited by lead ions. The E. coli enzyme crystallized in space group 1422 (unit cell dimensions a = b= 130.7 Å, c = 142.4 Å). The best crystals were obtained in the presence of the covalently bound inhibitor laevulinic acid. The yeast enzyme (expressed in E. coli) crystallized in the same space group (1422) but with a smaller unit cell volume (a = b = 103.7 Å, c = 167.7 Å). High resolution synchrotron data sets were obtained from both E. coli and yeast ALAD crystals by cryocooling to 100 K.
Surface topology of Minibody by selective chemical modifications and mass spectrometry
Authors: Zappacosta Francesca , Ingallinella Paolo , Scaloni Andrea , Pessi Antonello , Bianchi Elisabetta , Sollazzo Maurizio , Tramontano Anna , Marino Gennaro , Pucci Piero 
Published: February 2009

AbstractThe surface topology of the Minibody, a small de novo‐designed β‐protein, has been probed by a strategy that combines selective chemical modification with a variety of reagents and mass spectrometric analysis of the modified fragments. Under appropriate conditions, the susceptibility of individual residues primarily depends on their surface accessibility so that their relative reactivities can be correlated with their position in the tertiary structure of the protein. Moreover, this approach provides information on interacting residues, since intramolecular interactions might greatly affect the reactivity of individual side chains by altering their pKa values.The results of this study indicate that, while overall the Minibody model is correct, the β‐sheet formed by the N‐ and C‐terminal segments is most likely distorted. This is also in agreement with previous results that were obtained using a similar approach where mass spectrometry was used to identify Minibody fragments from limited proteolysis (Zappacosta F, Pessi A, Bianchi E, Venturini s, Sollazzo M, Tramontano A, Marino G, Pucci P. 1996. Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: The case of Minibody. Protein Sci 5902‐813). The chemical modification approach, in combination with limited proteolysis procedures, can provide useful, albeit partial, structural information to complement simulation techniques. This is especially valuable when, as in the Minibody case, an NMR and/or X‐ray structure cannot be obtained due to insufficient solubility of the molecule.
Identification of the epitopes of calcitonin gene‐related peptide (CGRP) for two anti‐CGRP monoclonal antibodies by 2D NMR
Authors: Hubbard JULIA A.M. , Raleigh Daniel P. , Bonnerjea Julian R. , Dobson Christopher M. 
Published: February 2009

AbstractThe interactions between calcitonin gene‐related peptide and FAB fragments prepared from two different high‐affinity anti‐CGRP monoclonal antibodies (CB3 and CD1) have been studied at physiological pH using the ability of 1H NMR to detect selectively regions of dynamic flexibility. The 37‐residue peptide retains considerable flexibility in regions of its sequence when bound to both antibodies; in each case, more than half of the residues can be seen to have linewidths little perturbed from those of the free peptide. However, the regions where substantial broadening of resonances occur, attributed to substantially reduced motional freedom of the peptide resulting from interactions within the antibody combining site, differ greatly in the two cases. In the complex with CB3 the results indicate that the restricted residues lie exclusively within the C‐terminal half of the peptide, and include residues 25 to 32 and the terminal two residues (36 and 37). By contrast, in the complex with CD1, the conformationally restricted residues appear to lie predominantly within the N‐terminal half of the CGRP molecule, particularly residues 4‐16, although several residues in the middle section of the sequence (22‐31) have reduced conformational freedom. These findings, consistent with the results from immunological assays, add considerably to our knowledge of the epitopes.
Empirical free energy calculation: Comparison to calorimetric data
Authors: Weng Zhiping , Delisi Charles , Vajda Sandor 
Published: February 2009

AbstractAn effective free energy potential, developed originally for binding free energy calculation, is compared to calorimetric data on protein unfolding, described by a linear combination of changes in polar and nonpolar surface areas. The potential consists of a molecular mechanics energy term calculated for a reference medium (vapor or nonpolar liquid), and empirical terms representing solvation and entropic effects. It is shown that, under suitable conditions, the free energy function agrees well with the calorimetric expression. An additional result of the comparison is an independent estimate of the side‐chain entropy loss, which is shown to agree with a structure‐based entropy scale. These findings confirm that simple functions can be used to estimate the free energy change in complex systems, and that a binding free energy evaluation model can describe the thermodynamics of protein unfolding correctly. Furthermore, it is shown that folding and binding leave the sum of solute‐solute and solute‐solvent van der Waals interactions nearly invariant and, due to this invariance, it may be advantageous to use a nonpolar liquid rather than vacuum as the reference medium.
The effect of Arg306 → Ala and Arg506 → Gln substitutions in the inactivation of recombinant human factor Va by activated protein C and protein S
Authors: Egan Jack O. , Kalafatis Michael , Mann Kenneth G. 
Published: February 2009

AbstractFactor Va (fVa) is inactivated by activated protein C (APC) by cleavage of the heavy chain at Arg306, Arg506, and Arg679. Site‐directed mutagenesis of human factor V cDNA was used to substitute Arg306 → Ala (rfVa306A) and Arg506 → Gln (rfVa506Q). Both the single and double mutants (rfVa306A/506Q) were constructed. The activation of these procofactors by α‐thrombin and their inactivation by APC were assessed in coagulation assays using factor V‐deficient plasma. All recombinant and wild‐type proteins had similar initial cofactor activity and identical activation products (a factor Va molecule composed of light and heavy chains). Inactivation of factor Va purified from human plasma (fVaPLASMA) in HBS Ca2+ +0.5% BSA or in conditioned media by APC in the presence of phospholipid vesicles resulted in identical inactivation profiles and displayed identical cleavage patterns. Recombinant wild‐type factor Va (rfVaWT) was inactivated by APC in the presence of phospholipid vesicles at an overall rate slower than fVaPLASMA. The rfVa306A and rfVa506Q mutants were each inactivated at rates slower than rfVaWT and fVaPLASMA. Following a 90‐min incubation with APC, rfVa306A and rfva506Q retain approximately 30‐40% of the initial cofactor activity. The double mutant, rfVa306A/506Q, was completely resistant to cleavage and inactivation by APC retaining 100% of the initial cofactor activity following a 90‐min incubation in the presence of APC. Recombinant maWT, rfVa306A, rfVa506Q, and rfVa306A/506Q were also used to evaluate the effect of protein S on the individual cleavage sites of the cofactor by APC. The initial rates of rfVaWT and rfVa306A inactivation in the presence of protein S were unchanged, indicating cleavage at Arg506 is not affected by protein S. The initial rate of rfVa506Q inactivation was increased, suggesting protein S slightly accelerates the cleavage at Arg30h. Over‐all, the data demonstrate high specificity with respect to cleavage sites for APC on factor Va and demonstrate that cleavages of the cofactor at both Arg306 and Arg506 are required for efficient factor Va inactivation.
Calcium binding to tandem repeats of EGF‐like modules. Expression and characterization of the EGF‐like modules of human Notch‐1 implicated in receptor‐ligand interactions
Authors: Rand Matthew D. , Lindblom Anders , Carlson Joyce , Villoutreix Bruno O. , Stenflo Johan 
Published: February 2009

AbstractThe Ca2+‐binding epidermal growth factor (cbEGF)‐like module is a structural component of numerous diverse proteins and occurs almost exclusively within repeated motifs. Notch‐1, a fundamental receptor for cell fate decisions, contains 36 extracellular EGF modules in tandem, of which 21 are potentially Ca2+‐binding. We report the Ca2+‐binding properties of EGF11‐12 and EGF10‐13 from human Notch‐1 (hNEGF11‐12 and hNEGF10‐13), modules previously shown to support Ca2+‐dependent interactions with the ligands Delta and Serrate. Ca2+ titrations in the presence of chromophoric chelators, 5,5′‐Br2BAPTA and 5‐NBAPTA, gave two binding constants for hNEGF11‐12, Kd1 = 3.4 × 10−5 M and Kd2 2.5 × 10−4 M. The high‐affinity site was found to be localized to hNEGF12. Titration of hNEGF10‐13 gave three binding constants, Kd1 = 3.1 × 10−6 M, Kd2 = 1.6 × 10−4 M, and Kd3 > 2.5 × 10−4 M, demonstrating that assembly of EGF modules in tandem can increase Ca2+ affinity. The highest affinity sites in hNEGF11‐12 and hNEGF10‐13 had 10 to 100‐fold higher affinity than reported for EGF32‐33 and EGF25‐31, respectively, from fibrillin‐1, a connective tissue protein with 43 cbEGF modules. A model of hNEGF11‐12 based on fibrillin‐1 EGF32‐33 demonstrates electronegative potential that could contribute to the higher affinity of the Ca2+‐binding site in hNEGF12. These data demonstrate that the Ca2+ affinity of cbEGF repeats can be highly variable among different classes of cbEGF containing proteins.
Unusual conformation of nicotinamide adenine dinucleotide (NAD) bound to diphtheria toxin: A comparison with NAD bound to the oxidoreductase enzymes
Authors: Bell Charles E. , Yeates Todd O. , Eisenberg David 
Published: February 2009

AbstractThe conformation of NAD bound to diphtheria toxin (DT), an ADP‐ribosylating enzyme, has been compared to the conformations of NAD(P) bound to 23 distinct NAD(P)‐binding oxidoreductase enzymes, whose structures are available in the Brookhaven Protein Data Bank. For the oxidoreductase enzymes, NAD(P) functions as a cofactor in electron transfer, whereas for DT, NAD is a labile substrate in which the N‐glycosidic bond between the nicotinamide ring and the N‐ribose is cleaved. All NAD(P) conformations were compared by (1) visual inspection of superimposed molecules, (2) RMSD of atomic positions, (3) principal component analysis, and (4) analysis of torsion angles and other conformational parameters. Whereas the majority of oxidoreductase‐bound NAD(P) conformations are found to be similar, the conformation of NAD bound to DT is found to be unusual. Distinctive features of the conformation of NAD bound to DT that may be relevant to DT's function as an ADP‐ribosylating enzyme include (1) an unusually short distance between the PN and N1N atoms, reflecting a highly folded conformation for the nicotinamide mononucleotide (NMN) portion of NAD, and (2) a torsion angle XN ∼ 0° about the scissile N‐glycosidic bond, placing the nicotinamide ring outside of the preferred anti and syn orientations. In NAD bound to DT, the highly folded NMN conformation and torsion angle XN ∼ 0° could contribute to catalysis, possibly by orienting the C1′N atom of NAD for nucleophilic attack, or by placing strain on the N‐glycosidic bond, which is cleaved by DT. The unusual overall conformation of NAD bound to DT is likely to reflect the structure of DT, which is unusual among NAD(P)‐binding enzymes. In DT, the NAD binding site is formed at the junction of two antiparallel β‐sheets. In contrast, although the 24 oxidoreductase enzymes belong to at least six different structural classes, almost all of them bind NAD(P) at the C‐terminal end of a parallel β‐sheet. The structural alignments and principal component analysis show that enzymes of the same structural class bind to particularly similar conformations of NAD(P), with few exceptions. The conformation of NAD bound to DT superimposes closely with that of an NAD analogue bound to Pseudomonas exotoxin A, an ADP‐ribosylating toxin that is structurally homologous to DT. This suggests that all of the ADP‐ribosylating enzymes that are structurally homologous to DT and ETA will bind a highly similar conformation of NAD.
Evaluating the energetics of empty cavities and internal mutations in proteins
Authors: Rashin Alexander A. , Rashin Bella H. , Rashin Annita , Abagyan Ruben 
Published: February 2009

AbstractThe energetics of cavity formation in proteins is evaluated with two different approaches and results are analyzed and compared to experimental data. In the first approach, free energy of cavity formation is extracted by RMS fitting from the distribution of numbers of cavities, N, with different volumes, Vcav' in 80 high‐resolution protein structures. It is assumed that the distribution of number of cavities according to their volume follows the Boltzmann law, NVcav = exp[(‐α.Vcav b)/kT], or its simplified form. Specific energy cost of cavity formation, α, extracted by RMS fitting from these distributions is compared to α values extracted from experimental free energies of cavity formation in T4 lysozyme fitted to similar expressions. It is found that fitting of both sets of data leads to similar magnitudes and uncertainties in the calculated free energy values. It is shown that Boltzmann‐like distribution of cavities can be derived for a simple model of an equilibrium interconversion between mutants in an extracellular system. We, however, suggest that a partitioning into cavity‐dependent and cavity‐independent terms may lose meaning when one attempts to describe mutation effects on protein stability in terms of specific free energy contributions. As an alternative approach, a direct molecular mechanics evaluation is attempted of T4 lysozyme destabilization by five single cavity‐creating mutations. The calculations are based on the approach used in calculations of the energetics of packing defects in crystals. For all mutations calculated destabilizations agree with the corresponding experimental values within +0.6 kcal/mol. A computational relaxation of the mutant was most difficult to achieve for the mutation producing the smallest cavity. However, calculations do not always reproduce crystallographically observed contraction/expansion of cavities. It is suggested that this may be related to usually observed large RMS differences (>1 Å) between crystallographic and energy‐minimized protein structures, and thus correct energetics might be easier to calculate than the correct geometry.