bluuebunny/crossref_metadata_2025_split huggingface dataset:
Crystal structure of p‐hydroxybenzoate hydroxylase reconstituted with the modified fad present in alcohol oxidase from methylotrophic yeasts: Evidence for an arabinoflavin
Authors: Van Berkel Willem J. H. , Eppink Michel H.M. , Schreuder Herman A. 
Published: February 2009

AbstractThe flavin prosthetic group (FAD) of p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens was replaced by a stereochemical analog, which is spontaneously formed from natural FAD in alcohol oxidases from methylotrophic yeasts. Reconstitution of p‐hydroxybenzoate hydroxylase from apoprotein and modified FAD is a rapid process complete within seconds. Crystals of the enzyme–substrate complex of modified FAD‐containing p‐hydroxybenzoate hydroxylase diffract to 2.1 Å resolution. The crystal structure provides direct evidence for the presence of an arabityl sugar chain in the modified form of FAD. The isoalloxazine ring of the arabinoflavin adenine dinucleotide (a‐FAD) is located in a cleft outside the active site as recently observed in several other p‐hydroxybenzoate hydroxylase complexes.Like the native enzyme, a‐FAD‐containing p‐hydroxybenzoate hydroxylase preferentially binds the phenolate form of the substrate (pKa = 7.2). The substrate acts as an effector highly stimulating the rate of enzyme reduction by NADPH (kred > 500 s−1). The oxidative part of the catalytic cycle of a‐FAD‐containing p‐hydroxybenzoate hydroxylase differs from native enzyme. Partial uncoupling of hydroxylation results in the formation of about 0.3 mol of 3,4‐dihydroxybenzoate and 0.7 mol of hydrogen peroxide per mol NADPH oxidized. It is proposed that flavin motion in p‐hydroxybenzoate hydroxylase is important for efficient reduction and that the flavin “out” conformation is associated with the oxidase activity.
The isomorphous structures of prethrombin2, hirugen–, and PPACK–thrombin: Changes accompanying activation and exosite binding to thrombin
Authors: Vijayalakshmi J. , Padmanabhan K.P. , Tulinsky A. , Mann K.G. 
Published: February 2009

AbstractThe X‐ray crystal structure of prethrombin2 (pre2), the immediate inactive precursor of α‐thrombin, has been determined at 2.0 Å resolution complexed with hirugen. The structure has been refined to a final R‐value of 0.169 using 14,211 observed reflections in the resolution range 8.0–2.0 Å. A total of 202 water molecules have also been located in the structure. Comparison with the hirugen–thrombin complex showed that, apart from the flexible beginning and terminal regions of the molecule, there are 4 polypeptide segments in pre2 differing in conformation from the active enzyme (Pro 186–Asp 194, Gly 216–Gly 223, Gly 142–Pro 152, and the Arg 15–Ile 16 cleavage region). The formation of the Ile 16–Asp 194 ion pair and the specificity pocket are characteristic of serine protease activation with the conformation of the catalytic triad being conserved.With the determination of isomorphous structures of hirugen–thrombin and d‐Phe‐Pro‐Arg chloromethyl ketone (PPACK)–thrombin, the changes that occur in the active site that affect the kinetics of chromogenic substrate hydrolysis on binding to the fibrinogen recognition exosite have been determined. The backbone of the Ala 190–Gly 197 segment in the active site has an average RMS difference of 0.55 Å between the 2 structures (about 3.7σ compared to the bulk structure). This segment has 2 type II β‐bends, the first bend showing the largest shift due to hirugen binding. Another important feature was the 2 different conformations of the side chain of Glu 192. The side chain extends to solvent in hirugen–thrombin, which is compatible with the binding of substrates having an acidic residue in the P3 position (protein‐C, thrombin platelet receptor). In PPACK–thrombin, the side chain of Asp 189 and the segment Arg 221A–Gly 223 move to provide space for the inhibitor, whereas in hirugenthrombin, the Ala 190–Gly 197 movement expands the active site region. Although 8 water molecules are expelled from the active site with PPACK binding, the inhibitor complex is resolvated with 5 other water molecules.
The structures of rnase a complexed with 3′‐CMP and d(CpA): Active site conformation and conserved water molecules
Authors: Zegers Ingrid , Maes Dominique , Dao‐Thi Minh‐Hoa , Wyns Lode , Poortmans Freddy , Palmer Rex 
Published: February 2009

AbstractThe interactions of RNase A with cytidine 3′‐monophosphate (3′‐CMP) and deoxycytidyl‐3′,5′‐deoxyadenosine (d(CpA)) were analyzed by X‐ray crystallography. The 3′‐CMP complex and the native structure were determined from trigonal crystals, and the d(CpA) complex from monoclinic crystals. The differences between the overall structures are concentrated in loop regions and are relatively small. The protein–inhibitor contacts are interpreted in terms of the catalytic mechanism. The general base His 12 interacts with the 2′ oxygen, as does the electrostatic catalyst Lys 41. The general acid His 119 has 2 conformations (A and B) in the native structure and is found in, respectively, the A and the B conformation in the d(CpA) and the 3′‐CMP complex. From the present structures and from a comparison with RNase T1, we propose that His 119 is active in the A conformation. The structure of the d(CpA) complex permits a detailed analysis of the downstream binding site, which includes His 119 and Asn 71. The comparison of the present RNase A structures with an inhibitor complex of RNase T1 shows that there are important similarities in the active sites of these 2 enzymes, despite the absence of any sequence homology. The water molecules were analyzed in order to identify conserved water sites. Seventeen water sites were found to be conserved in RNase A structures from 5 different space groups. It is proposed that 7 of those water molecules play a role in the binding of the N‐terminal helix to the rest of the protein and in the stabilization of the active site.
Residue–Residue contact substitution probabilities derived from aligned three‐dimensional structures and the identification of common folds
Authors: Rodionov Michael A. , Johnson Mark S. 
Published: February 2009

AbstractWe report the derivation of scores that are based on the analysis of residue‐residue contact matrices from 443 3‐dimensional structures aligned structurally as 96 families, which can be used to evaluate sequence‐structure matches. Residue–residue contacts and the more than 3 × 106 amino acid substitutions that take place between pairs of these contacts at aligned positions within each family of structures have been tabulated and segregated according to the solvent accessibility of the residues involved. Contact maps within a family of structures are shown to be highly conserved (∼75%) even when the sequence identity is approaching 10%. In a comparison involving a globin structure and the search of a sequence databank (>21,000 sequences), the contact probability scores are shown to provide a very powerful secondary screen for the top scoring sequence–structure matches, where between 69% and 84% of the unrelated matches are eliminated. The search of an aligned set of 2 globins against a sequence databank and the subsequent residue contact‐based evaluation of matches locates all 618 globin sequences before the first non‐globin match. From a single bacterial serine proteinase structure, the structural template approach coupled with residue–residue contact substitution data lead to the detection of the mammalian serine proteinase family among the top matches in the search of a sequence databank.
Disulfide crosslinks to probe the structure and flexibility of a designed four‐helix bundle protein
Authors: Regan Lynne , Rockwell Arlene , Wasserman Zelda , Degrado William 
Published: February 2009

AbstractThe introduction of disulfide crosslinks is a generally useful method by which to identify regions of a protein that are close together in space. Here we describe the use of disulfide crosslinks to investigate the structure and flexibility of a family of designed 4‐helix bundle proteins. The results of these analyses lend support to our working model of the proteins' structure and suggest that the proteins have limited main‐chain flexibility.
Topographic study of arrestin using differential chemical modifications and hydrogen/deuterium exchange
Authors: Ohguro Hiroshi , Palczewski Krzysztof , Walsh Kenneth A. , Johnson Richard S. 
Published: February 2009

AbstractArrestin is involved in the quenching of phototransduction by binding to photoactivated and phosphorylated rhodopsin (P‐Rho*). To study its conformational changes and regions interacting with P‐Rho*, arrestin was subjected to (1) differential acetylation at lysine residues in the presence and absence of P‐Rho*, and (2) amide hydrogen/deuterium exchange. Labeled protein was proteolysed and analyzed by mass spectrometry. Three Lys residues, 28, 176, and 211, were protected from acetylation in native arrestin, although they were not located in regions exhibiting slow amide hydrogen exchange rates. The presence of P‐Rho* protected lysine 201 from acetylation and partially protected 14 other lysyl residues, including (2, 5), (163, 166, 167), (232, 235, 236, 238), (267, 276), (298, 300), and 367, where parentheses indicate lysine residues found within the same peptide. In contrast, in the C‐terminal region of arrestin, lysyl residues (386, 392, 395) were more exposed upon binding to P‐Rho*. These data allowed us to identify functional regions in the arrestin molecule.
Crystallization and preliminary x‐ray crystallographic analysis of the 38‐kda immunodominant antigen of mycobacterium tuberculosis
Authors: Choudhary Abha , Vyas Meenakshi N. , Vyas Nand K. , Chang Zengyi , Quiocho Florante A. 
Published: February 2009

AbstractThe 38‐kDa lipoprotein is one of the most potent cell surface immunogens of Mycobacterium tuberculosis in antibody‐and T cell‐mediated reactions. Using a pure recombinant form of the protein, we have recently shown that it binds phosphate much like that of the phosphate‐binding protein (Mr = 34.4 kDa) that is localized in the periplasm of Escherichia coli and is involved as an initial receptor for active transport of phosphate. The purified 38‐kDa protein has been crystallized in 2 forms that are suitable for high‐resolution structural analyses. One form belongs to the monoclinic space group P21 with unit cell dimensions of a = 67.42 Å, b = 113.38 Å, c = 42.68 Å, and β = 108.53°. The other is of the orthorhombic space group P21212 with a = 125.46 Å, b = 72.27 Å, and c = 73.43 Å. Both crystal forms diffract to about 2 Å resolution on a fine focus rotating anode.
High mobility group protein, hmg‐1, contains insignificant glycosyl modification
Authors: Bernice Chao Y. , Betty Yan S. , Scovell William M. 
Published: February 2009

AbstractHigh mobility group protein‐1 (HMG‐1) is a ubiquitous, highly conserved, and abundant nuclear protein. Recent findings suggest that HMG‐1 may serve as a DNA chaperone protein and play a role in the regulation of transcription. There is a mounting interest in elucidating the mechanism by which HMG‐1 protein takes part in these activities. HMG‐1 has been reported to undergo an extensive array of posttranslational modifications, including glycosylation. We extend the earlier findings on the glycosylation of HMG‐1 by quantitating the amount of carbohydrate on HMG‐1 from calf thymus and chicken erythrocytes isolated by 2 different purification procedures. In addition, 2 different developmental stages (embryonic and adult) were examined in the chicken erythrocytes. The glycosyl composition was quantitated using the Dionex HPAE‐PAD II system. Furthermore, the presence of O‐linked GlcNAc on HMG‐1 was determined by the enzymatic incorporation of 3H‐galactose into HMG‐1 protein. Contrary to earlier reports, less than 0.5 mol of total monosaccharides (Fuc, Man, GalNH2, GlcNH2, Gal) were detected per mole of HMG‐1 protein, regardless of the source of the protein or the method of isolation. In addition, less than 0.002 mol of O‐linked GlcNAc per mole of HMG‐1 protein was detected. Thus, insignificant amount of glycosylation was found on HMG‐1 protein. Because O‐linked GlcNAc modification of proteins is believed to be a reversible posttranslational event, more definitive studies will need to be conducted before ruling out that the function of HMG‐1 protein is not regulated by glycosylation.
The subunit interfaces of oligomeric enzymes are conserved to a similar extent to the overall protein sequences
Authors: Grishin Nick V. , Phillips Margaret A. 
Published: February 2009

AbstractIt is well established that, within families of homologous enzymes, amino acid residues that are involved in the chemistry of the reaction are highly conserved. To determine if residues at the subunit interface of oligomeric enzymes with shared active sites are also conserved, comparative analysis of five enzyme families was undertaken. For the chosen enzyme families, sequence data were available for a large number of proteins and a three‐dimensional structure was known for at least two members of each family. The analysis indicates that the sub‐unit interface and the hydrophobic core of proteins from all five families have diverged to a similar extent to the overall protein sequences.
Interactions between hydrophobic side chains within α‐helices
Authors: Creamer Trevor P. , Rose George D. 
Published: February 2009

AbstractThe thermodynamic basis of helix stability in peptides and proteins is a topic of considerable interest. Accordingly, we have computed the interactions between side chains of all hydrophobic residue pairs and selected triples in a model helix, using Boltzmann‐weighted exhaustive modeling. Specifically, all possible pairs from the set Ala, Cys, His, Ile, Leu, Met, Phe, Trp, Tyr, and Val were modeled at spacings of (i, i + 2), (i, i + 3), and (i, i + 4) in the central turn of a model poly‐alanyl α‐helix. Significant interactions —both stabilizing and destabilizing —were found to occur at spacings of (i, i + 3) and (i, i + 4), particularly in side chains with rings (i.e., Phe, Tyr, Trp, and His). In addition, modeling of leucine triples in a helix showed that the free energy can exceed the sum of pairwise interactions in certain cases. Our calculated interaction values both rationalize recent experimental data and provide previously unavailable estimates of the constituent energies and entropies of interaction.
Thermodynamics of the temperature‐induced unfolding of globular proteins
Authors: Khechinashvili Nikolay N. , Janin Joël , Rodier Francis 
Published: February 2009

AbstractThe heat capacity, enthalpy, entropy, and Gibbs energy changes for the temperature‐induced unfolding of 11 globular proteins of known three‐dimensional structure have been obtained by microcalorimetric measurements. Their experimental values are compared to those we calculate from the change in solvent‐accessible surface area between the native proteins and the extended polypeptide chain. We use proportionality coefficients for the transfer (hydration) of aliphatic, aromatic, and polar groups from gas phase to aqueous solution, we estimate vibrational effects, and we discuss the temperature dependence of each constituent of the thermodynamic functions. At 25 °C, stabilization of the native state of a globular protein is largely due to two favorable terms: the entropy of non‐polar group hydration and the enthalpy of interactions within the protein. They compensate the unfavorable entropy change associated with these interactions (conformational entropy) and with vibrational effects. Due to the large heat capacity of nonpolar group hydration, its stabilizing contribution decreases quickly at higher temperatures, and the two unfavorable entropy terms take over, leading to temperature‐induced unfolding.
Simple models for the analysis of binding protein‐dependent transport systems
Authors: Shilton Brian H. , Mowbray Sherry L. 
Published: February 2009

AbstractMathematical modeling was used to evaluate experimental data for bacterial binding protein‐dependent transport systems. Two simple models were considered in which ligand‐free periplasmic binding protein interacts with the membrane‐bound components of transport. In one, this interaction was viewed as a competition with the ligand‐bound binding protein, whereas in the other, it was considered to be a consequence of the complexes formed during the transport process itself. Two sets of kinetic parameters were derived for each model that fit the available experimental results for the maltose system. By contrast, a model that omitted the interaction of ligand‐free binding protein did not fit the experimental data. Some applications of the successful models for the interpretation of existing mutant data are illustrated, as well as the possibilities of using mutant data to test the original models and sets of kinetic parameters. Practical suggestions are given for further experimental design.
Comparison of and limits of accuracy for statistical analyses of vibrational and electronic circular dichroism spectra in terms of correlations to and predictions of protein secondary structure
Authors: Pancoska Petr , Bitto Eduard , Janota Vit , Urbanova Marie , Gupta Vijai P. , Keiderling Timothy A. 
Published: February 2009

AbstractThis work provides a systematic comparison of vibrational CD (VCD) and electronic CD (ECD) methods for spectral prediction of secondary structure. The VCD and ECD data are simplified to a small set of spectral parameters using the principal component method of factor analysis (PC/FA). Regression fits of these parameters are made to the X‐ray‐determined fractional components (FC) of secondary structure. Predictive capability is determined by computing structures for proteins sequentially left out of the regression. All possible combinations of PC/FA spectral parameters (coefficients) were used to form a full set of restricted multiple regressions with the FC values, both independently for each spectral data set as well as for the two VCD sets and all the data grouped together. The complete search over all possible combinations of spectral parameters for different types of spectral data is a new feature of this study, and the focus on prediction is the strength of this approach. The PC/FA method was found to be stable in detail to expansion of the training set. Coupling amide II to amide I' parameters reduced the standard deviations of the VCD regression relationships, and combining VCD and ECD data led to the best fits. Prediction results had a minimum error when dependent on relatively few spectral coefficients. Such a limited dependence on spectral variation is the key finding of this work, which has ramifications for previous studies as well as suggests future directions for spectral analysis of structure. The best ECD prediction for helix and sheet uses only one parameter, the coefficient of the first subspectrum. With VCD, the best predictions sample coefficients of both the amide I' and II bands, but error is optimized using only a few coefficients. In this respect, ECD is more accurate than VCD for α‐helix, and the combined VCD (amide I'+II) predicts the β‐sheet component better than does ECD. Combining VCD and ECD data sets yields exceptionally good predictions by utilizing the strengths of each. However, the residual error, its distribution, and, most importantly, the lack of dependence of the method on many of the significant components derived from the spectra leads to the conclusion that the heterogeneity of protein structure is a fundamental limitation to the use of such spectral analysis methods. The underutilization of these data for prediction of secondary structure suggests spectral data could predict a more detailed descriptor.
The SEA module: A new extracellular domain associated with O‐glycosylation
Authors: Bork Peer , Patthy Laszlo 
Published: February 2009

AbstractUsing a variety of homology search methods and multiple alignments, a new extracellular module was identified in (1) agrin, (2) enterokinase, (3) a 63‐kDa sea urchin sperm protein, (4) perlecan, (5) the breast cancer marker MUC1 (episialin), (6) the cell surface antigen 114/A10, and (7/8) two functionally uncharacterized, probably extracellular, Caenorhabditis elegans proteins. Despite the functional diversity of these adhesive proteins, a common denominator seems to be their existence in heavily glycosylated environments. In addition, the better characterized proteins mentioned above contain all O‐glycosidiclinked carbohydrates such as heparan sulfate that contribute considerably to their molecular masses. The common module might regulate or assist binding to neighboring carbohydrate moieties.
Characterization of a new four‐chain coiled‐coil: Influence of chain length on stability
Authors: Fairman Robert , Chao Hann‐Guang , Mueller Luciano , Lavoie Thomas B. , Shen Liyang , Novotny Jiri , Matsueda Gary R. 
Published: February 2009

AbstractLimited information is available on inherent stabilities of four‐chain coiled‐coils. We have developed a model system to study this folding motif using synthetic peptides derived from sequences contained in the tetramerization domain of Lac repressor. These peptides are tetrameric as judged by both gel filtration and sedimentation equilibrium and the tetramers are fully helical as determined by CD. The four‐chain coiled‐coils are well folded as judged by the cooperativity of thermal unfolding and by the extent of dispersion in aliphatic chemical shifts seen in NMR spectra. In addition, we measured the chain length dependence of this four‐chain coiled‐coil. To this end, we developed a general procedure for nonlinear curve fitting of denaturation data in oligomeric systems. The dissociation constants for bundles that contain α‐helical chains 21, 28, and 35 amino acids in length are 3.1 × 10−12, 6.7 × 10−23, and 1.0 × 10−38 M3, respectively. This corresponds to tetramer stabilities (in terms of the peptide monomer concentration) of 180 μM, 51 nM, and 280 fM, respectively. Finally, we discuss the rules governing coiled‐coil formation in light of the work presented here.
Solution structure of the potassium channel inhibitor agitoxin 2: Caliper for probing channel geometry
Authors: Krezel Andrzej M. , Kasibhatla Chandrasekhar , Hidalgo Patricia , Mackinnon Roderick , Wagner Gerhard 
Published: February 2009

AbstractThe structure of the potassium channel blocker agitoxin 2 was solved by solution NMR methods. The structure consists of a triple‐stranded antiparallel β‐sheet and a single helix covering one face of the β‐sheet. The cysteine side chains connecting the β‐sheet and the helix form the core of the molecule. One edge of the β‐sheet and the adjacent face of the helix form the interface with the Shaker K+ channel. The fold of agitoxin is homologous to the previously determined folds of scorpion venom toxins. However, agitoxin 2 differs significantly from the other channel blockers in the specificity of its interactions. This study was thus focused on a precise characterization of the surface residues at the face of the protein interacting with the Shaker K+ channel. The rigid toxin molecule can be used to estimate dimensions of the potassium channel. Surface‐exposed residues, Arg24, Lys27, and Arg31 of the β‐sheet, have been identified from mutagenesis studies as functionally important for blocking the Shaker K+ channel. The sequential and spatial locations of Arg24 and Arg31 are not conserved among the homologous toxins. Knowledge on the details of the channel‐binding sites of agitoxin 2 formed a basis for site‐directed mutagenesis studies of the toxin and the K+ channel sequences. Observed interactions between mutated toxin and channel are being used to elucidate the channel structure and mechanisms of channel‐toxin interactions.