bluuebunny/crossref_metadata_2025_split huggingface dataset:
Crystal structures of bovine chymotrypsin and trypsin complexed to the inhibitor domain of alzheimer's amyloid β‐protein precursor (APPI) and basic pancreatic trypsin inhibitor (BPTI): Engineering of inhibitors with altered specificities
Authors: Scheidig Axel J. , Hynes Thomas R. , Pelletier Laura A. , Wells James A. , Kossiakoff Anthony A. 
Published: February 2009

AbstractThe crystal structures of the inhibitor domain of Alzheimer's amyloid β‐protein precursor (APPI) complexed to bovine chymotrypsin (C‐APPI) and trypsin (T‐APPI) and basic pancreatic trypsin inhibitor (BPTI) bound to chymotrypsin (C‐BPTI) have been solved and analyzed at 2.1 Å, 1.8 Å, and 2.6 Å resolution, respectively. APPI and BPTI belong to the Kunitz family of inhibitors, which is characterized by a distinctive tertiary fold with three conserved disulfide bonds. At the specificity‐determining site of these inhibitors (P1), residue 15(I)4 is an arginine in APPI and a lysine in BPTI, residue types that are counter to the chymotryptic hydrophobic specificity. In the chymotrypsin complexes, the Arg and Lys P1 side chains of the inhibitors adopt conformations that bend away from the bottom of the binding pocket to interact productively with elements of the binding pocket other than those observed for specificity‐matched P1 side chains. The stereochemistry of the nucleophilic hydroxyl of Ser 195 in chymotrypsin relative to the scissile P1 bond of the inhibitors is identical to that observed for these groups in the trypsin‐APPI complex, where Arg 15(I) is an optimal side chain for tryptic specificity. To further evaluate the diversity of sequences that can be accommodated by one of these inhibitors, APPI, we used phage display to randomly mutate residues 11, 13, 15, 17, and 19, which are major binding determinants. Inhibitor variants were selected that bound to either trypsin or chymotrypsin. As expected, trypsin specificity was principally directed by having a basic side chain at P1 (position 15); however, the P1 residues that were selected for chymotrypsin binding were His and Asn, rather than the expected large hydrophobic types. This can be rationalized by modeling these hydrophilic side chains to have similar H‐bonding interactions to those observed in the structures of the described complexes. The specificity, or lack thereof, for the other individual subsites is discussed in the context of the “allowed” residues determined from a phage display mutagenesis selection experiment.
Backbone makes a significant contribution to the electrostatics of α/β‐barrel proteins
Authors: Raychaudhuri Soumya , Younas Fayyaz , Karplus P. Andrew , Faerman Carlos H. , Ripoll Daniel R. 
Published: February 2009

AbstractThe electrostatic properties of seven α/β‐barrel enzymes selected from different evolutionary families were studied: triose phosphate isomerase, fructose‐1,6‐bisphosphate aldolase, pyruvate kinase, mandelate racemase, trimethylamine dehydrogenase, glycolate oxidase, and narbonin, a protein without any known enzymatic activity. The backbone of the α/β‐barrel has a distinct electrostatic field pattern, which is dipolar along the barrel axis. When the side chains are included in the calculations the general effect is to modulate the electrostatic pattern so that the electrostatic field is generally enhanced and is focused into a specific area near the active site. We use the electrostatic flux through a square surface near the active site to gauge the functionally relevant magnitude of the electrostatic field. The calculations reveal that in six out of the seven cases the backbone itself contributes greater than 45% of the total flux. The substantial electrostatic contribution of the backbone correlates with the known preference of α/β‐barrel enzymes for negatively charged substrates.
Solution structure of drosomycin, the first inducible antifungal protein from insects
Authors: Landon Céline , Sodano Patrick , Hetru Charles , Hoffmann Jules , Ptak Marius 
Published: February 2009

AbstractDrosomycin is the first antifungal protein characterized recently among the broad family of inducible peptides and proteins produced by insects to respond to bacterial or septic injuries. It is a small protein of 44 amino acid residues extracted from Drosophila melanogaster that exhibits a potent activity against filamentous fungi. Its three‐dimensional structure in aqueous solution was determined using 1H 2D NMR. This structure, involving an α‐helix and a twisted three‐stranded β‐sheet, is stabilized by three disulfide bridges. The corresponding Cysteine Stabilized αβ (CSαβ) motif, which was found in other defense proteins such as the antibacterial insect defensin A, short‐ and long‐chain scorpion toxins, as well as in plant thionins and potent antifungal plant defensins, appears as remarkably persistent along evolution.
Predicting protein secondary structure with probabilistic schemata of evolutionarily derived information
Authors: Thompson Michael J. , Goldstein Richard A. 
Published: February 2009

AbstractWe demonstrate the applicability of our previously developed Bayesian probabilistic approach for predicting residue solvent accessibility to the problem of predicting secondary structure. Using only single‐sequence data, this method achieves a three‐state accuracy of 67% over a database of 473 non‐homologous proteins. This approach is more amenable to inspection and less likely to overlearn specifics of a dataset than “black box” methods such as neural networks. It is also conceptually simpler and less computationally costly.We also introduce a novel method for representing and incorporating multiple‐sequence alignment information within the prediction algorithm, achieving 72% accuracy over a dataset of 304 non‐homologous proteins. This is accomplished by creating a statistical model of the evolutionarily derived correlations between patterns of amino acid substitution and local protein structure. This model consists of parameter vectors, termed “substitution schemata,” which probabilistically encode the structure‐based heterogeneity in the distributions of amino acid substitutions found in alignments of homologous proteins. The model is optimized for structure prediction by maximizing the mutual information between the set of schemata and the database of secondary structures.Unlike “expert heuristic” methods, this approach has been demonstrated to work well over large datasets. Unlike the opaque neural network algorithms, this approach is physicochemically intelligible. Moreover, the model optimization procedure, the formalism for predicting one‐dimensional structural features, and our previously developed method for tertiary structure recognition all share a common Bayesian probabilistic basis. This consistency starkly contrasts with the hybrid and ad hoc nature of methods that have dominated this field in recent years.
Reduced BPTI is collapsed. A pulsed field gradient NMR study of unfolded and partially folded bovine pancreatic trypsin inhibitor
Authors: Pan Hong , Barany George , Woodward Clare 
Published: February 2009

AbstractPulsed field gradient NMR was used to measure the hydrodynamic behavior of unfolded variants of bovine pancreatic trypsin inhibitor (BPTI). The unfolded BPTI species studied were [R]Abu, at pH 4.5 and pH 2.5, and unfolded [14‐38]Abu at pH 2.5. These were prepared by chemical synthesis. [R]Abu is a model for reduced BPTI; all cysteine residues are replaced by α‐amino‐n‐butyric acid (Abu). [14‐38]Abu retains cysteines 14 and 38, which form a disulfide bond, while the other cysteine residues are replaced by Abu. In the PFG experiments, the diffusion coefficient is measured as a function of protein concentration, and the value of D°—the diffusion coefficient extrapolated to infinite dilution—is determined. From D°, a value of the hydrodynamic radius, Rh, is computed from the Stokes‐Einstein relationship. At pH 4.5, [R]Abu has an Rh value significantly less than the value calculated for a random coil, while at pH 2.5 the experimental Rh value is the same as for a random coil. In view of the changes in NMR‐detected structure of [R]Abu at pH 4.5 versus pH 2.5 (Pan H, Barbar E, Barany G, Woodward C. 1995. Extensive non‐random structure in reduced and unfolded bovine pancreatic trypsin inhibitor. Biochemistry 34:13974‐13981), the collapse of reduced BPTI at pH 4.5 may be associated with the formation of non‐native hydrophobic clusters of pairs of side chains one to three amino acids apart in sequence. The diffusion constant of [14‐38]Abu was also measured at pH 4.5, where the protein is partially folded. An increase in hydrodynamic radius of partially folded [14‐38]Abu relative to native BPTI, is similar to the increase in radius of gyration measured for other proteins under “molten globule” conditions.
Structural studies on membrane‐embedded influenza hemagglutinin and its fragments
Authors: Gray Cameron , Tamm Lukas K. 
Published: February 2009

AbstractThe mechanism of influenza virus hemagglutinin (HA)‐mediated membrane fusion has been inferred in part from studies examining pH‐induced structural changes in soluble HA derivatives lacking the viral membrane anchor and, sometimes, the fusion peptide (the C‐ and N‐terminal residues of the HA2 chain, respectively). To reconcile structure‐based mechanisms of HA‐mediated membrane fusion with structural implications of functional studies performed on membrane‐embedded HA, we have undertaken attenuated total reflection Fourier transform infrared (ATR‐FTIR) spectroscopic analyses of membrane‐embedded HA (strain X:31) and its fragments reconstituted into supported lipid bilayers. The fragments correspond to proteolytic products with the majority of the HA1 chain and, in some cases, the fusion peptide removed (THA2 and THAF−2, respectively). In combination with R18 fluorescence dequenching to monitor the functional implications of HA, subunit removal, we have assessed the influence of pH and target membrane presentation on the secondary structures, orientations relative to the membrane, and dynamics of these molecules. We find that X:31 HA is more tilted towards the plane of the membrane under fusion than under resting conditions, that the tilting of HA depends on the presence of the HA1 chain, that the residues connecting the membrane‐inserted fusion peptide with the crystallographically determined coiled coil probably adopt an a‐helical conformation, and that several changes in the secondary structure and the amide H/D exchange kinetics occur as a result of acidification and target membrane presentation, which can be interpreted as small changes and a release of strain in the static and dynamic structure of membrane‐bound HA. THA2 mediates fusion, but less efficiently and with less pH‐selectivity than HA.
Ligand binding to proteins: The binding landscape model
Authors: Miller David W. , Dill Ken A. 
Published: February 2009

AbstractModels of ligand binding are often based on four assumptions: (1) steric fit: that binding is determined mainly by shape complementarity; (2) native binding: that ligands mainly bind to native states; (3) locality: that ligands perturb protein structures mainly at the binding site; and (4) continuity: that small changes in ligand or protein structure lead to small changes in binding affinity. Using a generalization of the 2D HP lattice model, we study ligand binding and explore these assumptions. We first validate the model by showing that it reproduces typical binding behaviors. We observe ligand‐induced denaturation, ANS and heme‐like binding, and “lock‐and‐key” and “induced‐fit” specific binding behaviors characterized by Michaelis‐Menten or more cooperative types of binding isotherms. We then explore cases where the model predicts violations of the standard assumptions. For example, very different binding modes can result from two ligands of identical shape. Ligands can sometimes bind highly denatured states more tightly than native states and yet have Michaelis‐Menten isotherms. Even low‐population binding to denatured states can cause changes in global stability, hydrogen‐exchange rates, and thermal B‐factors, contrary to expectations, but in agreement with experiments. We conclude that ligand binding, similar to protein folding, may be better described in terms of energy landscapes than in terms of simpler mass‐action models.
Oligomerization properties of GCN4 leucine zipper e and g position mutants
Authors: Zeng Xiangang , Zhu Hai , Lashel Hilal A. , Hu James C. 
Published: February 2009

AbstractPutative intersubunit electrostatic interactions between charged amino acids on the surfaces of the dimer interfaces of leucine zippers (g‐e' ion pairs) have been implicated as determinants of dimerization specificity. To evaluate the importance of these ionic interactions in determining the specificity of dimer formation, we constructed a pool of >65,000 GCN4 leucine zipper mutants in which all the e and g positions are occupied by different combinations of alanine, glutamic acid, lysine, or threonine. The oligomerization properties of these mutants were evaluated based on the phenotypes of cells expressing λ repressor‐leucine zipper fusion proteins. About 90% of the mutants do not form stable homooligomers. Surprisingly, approximately 8% of the mutant sequences have phenotypes consistent with the formation of higher‐order (>dimer) oligomers, which can be classified into three types based on sequence features. The oligomerization states of mutants from two of these types were determined by characterizing purified fusion proteins. The Type I mutant behaved as a tetramer under all tested conditions, whereas the Type III mutant formed a variety of higher‐order oligomers, depending on the solution conditions. Stable homodimers comprise less than 3% of the pool; several g‐e' positions in these mutants could form attractive ion pairs. Putative repulsive ion pairs are not found among the homodimeric mutants. However, patterns of charged residues at the e and g positions do not seem to be sufficient to predict either homodimer or heterodimer formation among the mutants.
The specificity of carboxypeptidase Y may be altered by changing the hydrophobicity of the S'1 binding pocket
Authors: Sørensen Steen Bech , Breddam Klaus 
Published: February 2009

AbstractThe S'1 binding pocket of carboxypeptidase Y is hydrophobic, spacious, and open to solvent, and the enzyme exhibits a preference for hydrophobic P'1 amino acid residues. Leu272 and Ser297, situated at the rim of the pocket, and Leu267, slightly further away, have been substituted by site‐directed mutagenesis. The mutant enzymes have been characterized kinetically with respect to their P'1 substrate preferences using the substrate series FA‐Ala‐Xaa‐OH (Xaa = Leu, Glu, Lys, or Arg) and FA‐Phe‐Xaa‐OH (Xaa = Ala, Val, or Leu). The results reveal that hydrophobic P'1 residues bind in the vicinity of residue 272 while positively charged P'1 residues interact with Ser297. Introduction of Asp or Glu at position 267 greatly reduced the activity toward hydrophobic P'1 residues (Leu) and increased the activity two‐ to three‐fold for the hydrolysis of substrates with Lys or Arg in P'1. Negatively charged substituents at position 272 reduced the activity toward hydrophobic P'1 residues even more, but without increasing the activity toward positively charged P'1 residues. The mutant enzyme L267D+L272D was found to have a preference for substrates with C‐terminal basic amino acid residues. The opposite situation, where the positively charged Lys or Arg were introduced at one of the positions 267, 272, or 297, did not increase the rather low activity toward substrates with Glu in the P'1 position but greatly reduced the activity toward substrates with C‐terminal Lys or Arg due to electrostatic repulsion. The characterized mutant enzymes exhibit various specificities, which may be useful in C‐terminal amino acid sequence determinations.
Urea‐induced conformational changes in cold‐ and heat‐denatured states of a protein, Streptomyces subtilisin inhibitor
Authors: Konno Takashi , Kamatari Yuji O. , Kataoka Mikio , Akasaka Kazuyuki 
Published: February 2009

AbstractStreptomyces subtilisin inhibitor (SSI) is known to exist in at least two distinct denatured states, cold‐denatured (D') and heat‐denatured (D) under acidic conditions. In the present work, we investigated the manner how increasing urea concentration from 0 to 8 M changes the polypeptide chain conformation of SSI that exists initially in the D' and D states as well as in the native state (N), in terms of the secondary structure, the tertiary structure, and the chain form, based on the results of the experiments using circular dichroism (CD), small‐angle X‐ray scattering (SAXS) and 1H‐NMR spectroscopy. Our results indicate that the urea‐induced conformational transitions of SSI under typical conditions of D' (pH 1.8, 3°C) occur at least in two steps. In the urea concentration range of 0‐2 M (step 1), a cooperative destruction of the tertiary structure occurs, resulting in a mildly denatured state (DU), which may still contain a little amount of secondary structures. In the concentration range of 2‐4 M urea (step 2), the DU state gradually loses its residual secondary structure, and increases the radius of gyration nearly to a maximum value. At 4 M urea, the polypeptide chain is highly disordered with highly mobile side chains. Increasing the urea concentration up to 8 M probably results in the more highly denatured or alternatively the stiffer chain conformations. The conformational transition starting from the N state proceeds essentially the same way as in the above scheme in which D' is replaced with N. The conformational transition starting from the D state lacks step 1 because the D state contains no tertiary structures and is similar to the DU state. The fact that similar conformations are reached at urea concentrations above 2 M from different conformations of D', D, and N indicates that the effect of urea dominates in determining the polypeptide conformation of SSI in the denatured states rather than the pH and temperature.
1H, 13C, and 15N backbone assignment and secondary structure of the receptor‐binding domain of vascular endothelial growth factor
Authors: Fairbrother Wayne J. , Champe Mark A. , Christinger Hans W. , Keyt Bruce A. , Starovasnik Melissa A. 
Published: February 2009

AbstractNearly complete sequence‐specific 1H, 13C, and 15N resonance assignments are reported for the backbone atoms of the receptor‐binding domain of vascular endothelial growth factor (VEGF), a 23‐kDa homodimeric protein that is a major regulator of both normal and pathological angiogenesis. The assignment strategy relied on the use of seven 3D triple‐resonance experiments [HN(CO)CA, HNCA, HNCO, (HCA)CONH, HN(COCA)HA, HN(CA)HA, and CBCA(CO)NH] and a 3D 15N‐TOCSY‐HSQC experiment recorded on a 0.5 mM (12 mg/mL) sample at 500 MHz, pH 7.0, 45°C. Under these conditions, 15N relaxation data show that the protein has a rotational correlation time of 15.0 ns. Despite this unusually long correlation time, assignments were obtained for 94 of the 99 residues; 8 residues lack amide 1H and 15N assignments, presumably due to rapid exchange of the amide 1H with solvent under the experimental conditions used. The secondary structure of the protein was deduced from the chemical shift indices of the 1Hα, 13Cα, 13Cβ, and 13CO nuclei, and from analysis of backbone NOEs observed in a 3D 15N‐NOESY‐HSQC spectrum. Two helices and a significant amount of β‐sheet structure were identified, in general agreement with the secondary structure found in a recently determined crystal structure of a similar VEGF construct [Muller YA et al., 1997, Proc Natl Acad Sci USA 94:7192‐7197].
Cloning, expression, and crystallization of a hyperthermophilic protein that is homologous to the eukaryotic translation initiation factor, eIF5A
Authors: Kim Kyeong Kyu , Yokota Hisao , Kim Rosalind , Kim Sung‐Hou 
Published: February 2009

AbstractA gene coding for a protein homologous to a translation initiation factor of eukaryotes, eIF5A, was cloned from Methanococcus jannaschii, a hyperthermophile with an optimum growth temperature of 85 °C. The protein was overexpressed, purified and crystallized. The crystals were obtained by vapor diffusion method with 8% PEG 4000 as precipitant and belong to space group P4122 with unit cell dimensions a = b = 45.52 Å and c = 155.59 Å. These crystals diffract to at least 2.2 Å resolution.
Structure of a pancreatic α‐amylase bound to a substrate analogue at 2.03 Å resolution
Authors: Qian Minxie , Spinelli Silvia , Payan FrançOise , Driguez Hugues 
Published: February 2009

AbstractThe structure of pig pancreatic α‐amylase in complex with carbohydrate inhibitor and proteinaceous inhibitors is known but the successive events occurring at the catalytic center still remain to be elucidated. The X‐ray structure analysis of a crystal of pig pancreatic α‐amylase (PPA, EC 3.2.1.1.) soaked with an enzyme‐resistant substrate analogue, methyl 4,4′‐dithio‐α‐maltotrioside, showed electron density corresponding to the binding of substrate analogue molecules at the active site and at the “second binding site.” The electron density observed at the active site was interpreted in terms of overlapping networks of oligosaccharides, which show binding of substrate analogue molecules at subsites prior to and subsequent to the cleavage site. A weaker patch of density observed at subsite ‐1 (using a nomenclature where the site of hydrolysis is taken to be between subsites ‐1 and +1) was modeled with water molecules. Conformational changes take place upon substrate analogue binding and the “flexible loop” that constitutes the surface edge of the active site is observed in a specific conformation. This confirms that this loop plays an important role in the recognition and binding of the ligand. The crystal structure was refined at 2.03 Å resolution, to an R‐factor of 16.0 (Rfree 18.5).
Oxidation of apolipoprotein(a) inhibits kringle‐associated lysine binding: The loss of intrinsic protein fluorescence suggests a role for tryptophan residues in the lysine binding site
Authors: Hermann Anita , Laws William R. , Harpel Peter C. 
Published: February 2009

AbstractLipoprotein(a) [Lp(a)] is a low‐density lipoprotein complex consisting of apolipoprotein(a) [apo(a)] disulfide‐linked to apolipoprotein B‐100. Lp(a) has been implicated in atherogenesis and thrombosis through the lysine binding site (LBS) affinity of its kringle domains. We have examined the oxidative effect of 2,2′‐azobis‐(amidinopropane) HCI (AAPH), a mild hydrophilic free radical initiator, upon the ability of Lp(a) and recombinant apo(a), r‐apo(a), to bind through their LBS domains. AAPH treatment caused a time‐dependent decrease in the number of functional Lp(a) or r‐apo(a) molecules capable of binding to fibrin or lysine‐Sepharose and in the intrinsic protein fluorescence of both Lp(a) and r‐apo(a). The presence of a lysine analogue during the reaction prevented the loss of lysine binding and provided a partial protection from the loss of tryptophan fluorescence. The partial protection of fluorescence by lysine analogues was observed in other kringle‐containing proteins, but not in proteins lacking kringles. No significant aggregation, fragmentation, or change in conformation of Lp(a) or r‐apo(a) was observed as assessed by native or SDS‐PAGE, light scattering, retention of antigenicity, and protein fluorescence emission spectra. Our results suggest that AAPH destroys amino acids in the kringles of apo(a) that are essential for lysine binding, including one or more tryptophan residues. The present study, therefore, raises the possibility that the biological roles of Lp(a) may be mediated by its state of oxidation, especially in light of our previous study showing that the reductive properties of sulfhydryl‐containing compounds increase the LBS affinity of Lp(a) for fibrin.