bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interaction of phlorizin, a potent inhibitor of the Na+/D‐glucose cotransporter, with the NADPH‐binding site of mammalian catalases
Authors: Kitlar Tomas , Döring Frank , Kinne Rolf K.H. , Deutscher Josef , Diedrich Donald F. , Frank Rainer , Wallmeier Holger 
Published: July 2010

AbstractPhlorizin is a reversible inhibitor of the renal and small intestinal Na+/D‐glucose cotransporter. In an attempt to purify the Na+/D‐glucose cotransporter from a pig kidney brush border membrane fraction, we used an Affi‐Gel affinity chromatography column to which 3‐aminophlorizin had been coupled. A protein, composed according to crosslinking experiments of at least 3 subunits of molecular weight 60 kDa, was found to bind specifically to the phlorizin column. This protein was subsequently identified as catalase by sequence homology of three of its tryptic fragments to the sequence of several mammalian catalases as well as by its enzymatic activity. Although bovine liver catalase was bound tightly to the affinity matrix, phlorizin had no effect on the ability of the enzyme to degrade H2O2. In contrast, the Aspergillus niger and Neurospora crussa catalases did not bind to the phlorizin column. This difference may be related to the fact that mammalian catalases, but not the fungal catalases, contain an NADPH binding site with a yet unknown function. Interestingly, bovine liver catalase could be eluted with 50 μM NADPH from phlorizin columns. Irradiation in the presence of [3H]4‐azidophlorizin allowed photolabeling of bovine liver catalase, which was prevented by the presence of 10 μM NADPH. After digestion of photolabeled catalase with chymotrypsin, a radioactive peptide was detected that was absent in catalase protected with NADPH. Docking simulations suggested that phlorizin can bind to the NADPH binding site with high affinity.
A structural basis for the interaction of urea with lysozyme
Authors: Pike Ashley C.W. , Acharya K. Ravi 
Published: July 2010

AbstractThe effect of urea on the crystal structure of hen eggwhite lysozyme has been investigated using X‐ray crystallography. High resolution structures have been determined from crystals grown in the presence of 0, 0.7, 2, 3, 4, and 5 M urea and from crystals soaked in 9 M urea. All the forms are essentially isomorphous with the native type I1 crystals, and the derived structures exhibit excellent geometry and RMS differences from ideality in bond distances and angles. Comparison of the urea complex structures with the native enzyme (type II form, at 1.5 Å resolution) indicates that the effect of urea is minimal over the concentration range studied. The mean difference in backbone conformation between the native enzyme and its urea complexes varies from 0.18 to 0.49 Å. Conformational changes are limited to flexible surface loops (Thr 69‐Asn 74, Ser 100‐Asn 103), the active site loop (Asn 59‐Cys go), and the C‐terminus (Cys 127‐Leu 129). Urea molecules are bound to distinct sites on the surface of the protein. One molecule is bound to the active site cleft's C subsite, at all concentrations, in a fashion analogous to that of the N‐acetyl substituent of substrate and inhibitor sugars normally bound to this site. Occupation of this subsite by urea alone does not appear to induce the conformational changes associated with inhibitor binding.
A novel zinc‐binding motif found in two ubiquitous deaminase families
Authors: Reizer Jonathan , Buskirk Stewart , Reizer Aiala , Saier Milton H. , Bairoch Amos 
Published: July 2010

AbstractTwo families of deaminases, one specific for cytidine, the other for deoxycytidylate, are shown to possess a novel zinc‐binding motif, here designated ZBS. We have (1) identified the protein members of these 2 families, (2) carried out sequence analyses that allow specification of this zinc‐binding motif, and (3) determined signature sequences that will allow identification of additional members of these families as their sequences become available.
Thermodynamics of staphylococcal nuclease denaturation. II. The A‐state
Authors: Carra John H. , Anderson Elizabeth A. , Privalov Peter L. 
Published: July 2010

AbstractStaphylococcal nuclease, at low pH and in the presence of high salt concentrations, has previously been proposed to exist in a partially folded or molten globule form called the “A‐state” (Fink et al., 1993, Protein Sci 2:1155–1160). We have found that the A‐state of nuclease at pH 2.1 in the presence of moderate to high salt concentrations and at low temperature exists in a substantially folded form structurally more similar to a native state. The A‐state has the far‐UV circular dichroism spectra characteristic of the native protein, which indicates that it has a large degree of secondary structure. Upon heating, the A‐state denatures with a sigmoidal change in far‐UV ellipticity and an observable peak in a differential scanning calorimeter trace, indicating that it is thermodynamically distinct from the denatured state. Three different mutations in a residue normally buried in the protein's core stabilize or destabilize the A‐state in the same way as they affect the denaturation of the native state. The A‐state must, therefore, contain at least some tertiary packing of side chains. Unlike the native state, which shows cold denaturation at low temperatures, the A‐state is most stable at temperatures below 0°C.
Crystallization and preliminary structural studies of neurotrophin‐3
Authors: Kelly Jennifer A. , Singer Elizabeth , Osslund Timothy D. , Yeates Todd O. 
Published: July 2010

AbstractNeurotrophin‐3 (NT‐3) has been crystallized in 2 forms. Orthorhombic crystals, space group P21212, diffracted to 2.8 Å and have cell dimensions a = 39.1 Å, b = 54.0 Å, and c = 65.5 Å. The second form is space group P43212, with cell dimensions a = b = 67.1 Å, and c = 107.9 Å. The tetragonal crystals diffract to 2.8 Å at room temperature and 2.5 Å at −100 °C. The unit cell dimensions change significantly upon freezing, a = b = 66.1 Å, and c = 102.8 Å. Phases for the orthorhombic form were obtained by molecular replacement using nerve growth factor as the search model. A partially refined model of the NT‐3 dimer (75% complete) was then oriented and positioned in the tetragonal cell.
Conformational stability of HPr: The histidine‐containing phosphocarrier protein from Bacillus subtilis
Authors: Martin Scholtz J. 
Published: July 2010

AbstractThe conformational stability of the histidine‐containing phosphocarrier protein (HPr) from Bacillus subtilis has been determined using a combination of thermal unfolding and solvent denaturation experiments. The urea‐induced denaturation of HPr was monitored spectroscopically at fixed temperatures and thermal unfolding was performed in the presence of fixed concentrations of urea. These data were analyzed in several different ways to afford a measure of the cardinal parameters (ΔHg, Tg, ΔSg, and ΔCp) that describe the thermodynamics of folding for HPr. The method of Pace and Laurents (Pace CN, Laurents DV, 1989, Biochemistry 28:2520–2525) was used to estimate ΔCP as was a global analysis of the thermal‐ and urea‐induced unfolding data. Each method used to analyze the data gives a similar value for ΔCp (1,170 Δ 50 cal mol−1 K−1). Despite the high melting temperature for HPr (Tg = 73.5 °C), the maximum stability of the protein, which occurs at 26 °C, is quite modest (AGs = 4.2 kcal mol−1). In the presence of moderate concentrations of urea, HPr exhibits cold denaturation, and thus a complete stability curve for HPr, including a measure of ΔCp, can be achieved using the method of Chen and Schellman (Chen B, Schellman JA, 1989, Biochemistry 28:685–691). A comparison of the different methods for the analysis of solvent denaturation curves is provided and the effects of urea on the thermal stability of this small globular protein are discussed. The methods presented will be of general utility in the characterization of the stability curve for many small proteins.
Crystallographic study of the structure of colipase and of the interaction with pancreatic lipase
Authors: Egloff Marie‐Pierre , Sarda Louis , Verger Robert , Cambillau Christian , Tilbeurgh Herman Van 
Published: July 2010

AbstractColipase (Mr 10 kDa) confers catalytic activity to pancreatic lipase under physiological conditions (high bile salt concentrations). Previously determined 3‐Å‐resolution X‐ray structures of lipase‐colipase complexes have shown that, in the absence of substrate, colipase binds to the noncatalytic C‐terminal domain of pancreatic lipase (van Tilbeurgh H, Sarda L, Verger R, Cambillau C, 1992, Nature 559:159–162; van Tilbeurgh et al., 1993a, Nature 362:814–820). Upon lipid binding, conformational changes at the active site of pancreatic lipase bring a surface loop (the lid) in contact with colipase, creating a second binding site for this cofactor. Covalent inhibition of the pancreatic lipase by a phosphonate inhibitor yields better diffracting crystals of the lipase‐colipase complex. From the 2.4‐Å‐resolution structure of this complex, we give an accurate description of the colipase. It confirms the previous proposed disulfide connections (van Tilbeurgh H, Sarda L, Verger R, Cambillau C, 1992, Nature 359:159–162; van Tilbeurgh et al., 1993a, Nature 362:814–820) that were in disagreement with the biochemical assignment (Chaillan C, Kerfelec B, Foglizzo E, Chapus C, 1992, Biochem Biophys Res Commun 184:206–211). Colipase lacks well‐defined secondary structure elements. This small protein seems to be stabilized mainly by an extended network of five disulfide bridges that runs throughout the flatly shaped molecule, reticulating its four finger‐like loops. The colipase surface can be divided into a rather hydrophilic part, interacting with lipase, and a more hydrophobic part, formed by the tips of the fingers. The interaction between colipase and the C‐terminal domain of lipase is stabilized by eight hydrogen bonds and about 80 van der Waals contacts. Upon opening of the lid, three more hydrogen bonds and about 28 van der Waals contacts are added, explaining the higher apparent affinity in the presence of a lipid/water interface. The tips of the fingers are very mobile and constitute the lipid interaction surface. Two detergent molecules that interact with colipase were observed in the crystal, covering part of the hydrophobic surface.
Identification of an essential acidic residue in Cdc25 protein phosphatase and a general three‐dimensional model for a core region in protein phosphatases
Authors: Eckstein Jens W. , Beer‐Romero Peggy , Berdo Ingrid 
Published: July 2010

AbstractThe reaction mechanism of protein tyrosine phosphatases (PTPases) and dual‐specificity protein phosphatases is thought to involve a catalytic aspartic acid residue. This residue was recently identified by site‐directed mutagenesis in Yersinia PTPase, VHR protein phosphatase, and bovine low molecular weight protein phosphatase. Herein we identify aspartic acid 383 as a potential candidate for the catalytic acid in human Cdc25A protein phosphatase, using sequence alignment, structural information, and site‐directed mutagenesis. The D383N mutant enzyme exhibits a 150‐fold reduction in kcat, with Km only slightly changed. Analysis of sequence homologies between several members of the Cdc25 family and deletion mutagenesis substantiate the concept of a two‐domain structure for Cdc25, with a regulatory N‐terminal and a catalytic C‐terminal domain. Based on the alignment of catalytic residues and secondary structure elements, we present a three‐dimensional model for the core region of Cdc25. By comparing this three‐dimensional model to the crystal structures of PTP1b, Yersinia PTPase, and bovine low molecular weight PTPase, which share only very limited amino acid sequence similarities, we identify a general architecture of the protein phosphatase core region, encompassing the active site loop motif HCXXXXXR and the catalytic aspartic acid residue.
Structure in solution of a four‐helix lipid binding protein
Authors: Heinemann Bo , Andersen Kim Vilbour , Nielsen Peter Reinholt , Bech Lene Mølskov , Poulsen Flemming M. 
Published: July 2010

AbstractBecause of the low solubility of lipids in water, intercellular and intracellular pathways of lipid transfer are necessary, e.g., for membrane formation. The mechanism by which lipids in vivo are transported from their site of biogenesis (endoplasmatic reticulum and the chloroplasts) to their place of action is unknown. Several small plant proteins with the ability to mediate transfer of radiolabeled phospholipids in vitro from liposomal donor membranes to mitochondrial and chloroplast acceptor membranes have been isolated, and a protein with this ability, the nonspecific lipid transfer protein (nsLTP) isolated from barley seeds (bLTP), has been studied here. The structure and the protein lipid interactions of lipid transfer proteins are relevant for the understanding of their function, and here we present the three‐dimensional structure in solution of bLTP as determined by NMR spectroscopy. The 1H NMR spectrum of the 91‐residue protein was assigned for more than 97% of the protein 1H atoms, and the structure was calculated on the basis of 813 distance restraints from 1H‐1H nuclear Overhauser effects, four disulfide bond restraints, from dihedral angle restraints for 66 ø‐angles, 61 χ1 angles, and 2 χ2 angles, and from 31 sets of hydrogen bond restraints. The solution structure of bLTP consists of four well‐defined α‐helices A‐D (A, Cys 3‐Gly 19; B, Gly 25‐Ala 38; C, Arg 44‐Gly 57; D, Leu 63‐Cys 73), separated by three short loops that are less well defined and concluded by a well defined C‐terminal peptide segment with no observable regular secondary structure. For the 17 structures that are used to represent the solution structure of bLTP, the RMS deviation to an average structure is 0.63 Å 0.04 Å for backbone atoms and 0.93 Å± 0.06 Å for all heavy atoms. The secondary structure elements and their locations in the sequence resemble those of nsLTP from two other plant species, wheat and maize, whose structures were previously determined (Gincel E et al, 1995, Eur J Biochem 226:413–422; Shin DH et al, 1995, Structure 3:189–199). In bLTP, the residues analogous to those in maize nsLTP that constitute the palmitate binding site are forming a similar hydrophobic cavity and a potential acyl group binding site. Analysis of the solution structure of bLTP and bLTP in complex with a ligand might provide information on the conformational changes in the protein upon ligand binding and subsequently provide information on the mode of ligand uptake and release. In this work, we hope to establish a foundation for further work of determining the solution structure of bLTP in complex with palmitoyl coenzyme A, which is a suitable ligand, and subsequently to outline the mode of ligand binding.
Interaction of partially unfolded forms ofTorpedoacetylcholinesterase with liposomes
Authors: Shin Irina , Silman Israel , Weiner Lev M. 
Published: July 2010

AbstractA water‐soluble dimeric form of acetylcholinesterase from electric organ tissue ofTorpedo californicawas obtained by solubilization with phosphatidylinositol‐specific phospholipase C of the glycophosphatidylinositol‐anchored species, followed by purification by affinity chromatography. The water‐soluble species, in its catalytically active native conformation, did not interact with unilamellar vesicles of dimyristoylphosphatidylcholine. We previously showed that either chemical modification or exposure to low concentrations of guanidine hydrochloride converted the native enzyme to compact, partially unfolded species with the physicochemical characteristics of the molten globule state. In the present study, it was shown that such molten globule species, whether produced by mild denaturation or by chemical modification, interacted efficiently with small unilamellar vesicles. Binding was not accompanied by significant vesicle fusion, but transient leakage occurred at the time of binding. The bound acetylcholinesterase reduced the transition temperature of the vesicles slightly, and NMR data suggested that it interacted primarily with the head‐group region of the bilayer. The effects of tryptic digestion of the bound acetylcholinesterase were monitored by gel electrophoresis under denaturing conditions. It was found that a single polypeptide, of mass ∼5 kDa, remained associated with the vesicles. Sequencing revealed that this is a tryptic peptide corresponding to the sequence Glu 268‐Lys 315. This polypeptide contains the longest hydrophobic sequence in the protein, Leu 274‐Met 308, as identified on the basis of hydropathy plots. Inspection of the three‐dimensional structure of acetylcholinesterase reveals that this hydrophobic sequence is largely devoid of tertiary structure and is localized primarily on the surface of the protein. It is suggested that this hydrophobic sequence is aligned parallel to the surface of the vesicle membrane, with nonpolar residues undergoing shallow penetration into the bilayer.
A structural basis for enantioselective inhibition of Candida rugosa lipase by long‐chain aliphatic alcohols
Authors: Holmquist Mats , Hæffner Fredrik , Norin Torbjörn , Hult Karl 
Published: July 2010

AbstractMolecular modeling showed that the enantiomers of heptyl 2‐methyldecanoate are productively bound to the active site of Candida rugosa lipase in quite different conformations. The fast‐reacting S‐enantiomer may well occupy the previously identified acyl‐binding tunnel in the active site of the lipase. By contrast, the slow‐reacting R‐enantiomer must be bound to the active site, leaving the tunnel empty to allow the formation of two catalytically essential hydrogen bonds between His 449 of the catalytic triad and the transition state of the catalyzed reaction. This information enables us to propose a molecular mechanism explaining how long‐chain aliphatic alcohols act as enantioselective inhibitors of this lipase in the resolution of 2‐methyldecanoic acid. Long‐chain aliphatic alcohols may coordinate to the acyl‐binding tunnel of the C. rugosa lipase, thereby selectively inhibiting the turnover of the fast‐reacting S‐enantiomer, thus resulting in a lowered enantioselectivity in the resolution.
Improving protein secondary structure prediction with aligned homologous sequences
Authors: Di Francesco V. , Garnier J. , Munson P.J. 
Published: July 2010

AbstractMost recent protein secondary structure prediction methods use sequence alignments to improve the prediction quality. We investigate the relationship between the location of secondary structural elements, gaps, and variable residue positions in multiple sequence alignments. We further investigate how these relationships compare with those found in structurally aligned protein families. We show how such associations may be used to improve the quality of prediction of the secondary structure elements, using the Quadratic‐Logistic method with profiles. Furthermore, we analyze the extent to which the number of homologous sequences influences the quality of prediction. The analysis of variable residue positions shows that surprisingly, helical regions exhibit greater variability than do coil regions, which are generally thought to be the most common secondary structure elements in loops. However, the correlation between variability and the presence of helices does not significantly improve prediction quality. Gaps are a distinct signal for coil regions. Increasing the coil propensity for those residues occurring in gap regions enhances the overall prediction quality. Prediction accuracy increases initially with the number of homologues, but changes negligibly as the number of homologues exceeds about 14. The alignment quality affects the prediction more than other factors, hence a careful selection and alignment of even a small number of homologues can lead to significant improvements in prediction accuracy.
Solvent isotope effects on the refolding kinetics of hen egg‐white lysozyme
Authors: Itzhaki Laura S. , Evans Philip A. 
Published: July 2010

AbstractSolvent isotope effects have been observed on the in vitro refolding kinetics of a protein, hen lysozyme. The rates of two distinct phases of refolding resolved by intrinsic fluorescence have been found to be altered, to differing extents, in D2O compared with H2O, and experiments have been conducted aimed at assessing the contributions to these effects from various possible sources. The rates were found to be essentially independent of whether backbone amide nitrogens were protiated or deuterated, indicating that making and breaking of their hydrogen bonding interactions is not associated with a substantial isotope effect. Neither were the rates significantly affected by adding moderate concentrations of sucrose or glycerol to the refolding buffer, suggesting that viscosity differences between H2O and D2O are also unlikely to explain the isotope effects. The data suggest that different factors, acting in opposing directions, may be dominant under different conditions. Thus, the isotope effect on the rate‐determining step was found to be qualitatively reversed on going to low pH, suggesting that one component is probably associated with changes in the environments of carboxylate groups in forming the folding transition state. This term disappears at low pH as these groups are protonated and an opposing effect then dominates. It was not possible to identify this other effect on the basis of the present data, but a dependence of the hydrophobic interaction on solvent isotopic composition is a likely candidate.
The protein machinery of vesicle budding and fusion
Authors: Rothman James E. 
Published: July 2010

AbstractA general protein machinery that buds and fuses transport vesicles is harnessed to generate the complex web of intracellular transport pathways critical for such diverse processes as cell growth, endocytosis, hormone release, and neurotransmission. With this appreciation, the challenge of understanding the precise molecular mechanisms of these many facets of cell biology has been reduced to a series of problems in protein structure and chemistry.
Crystallographic determination of the structures of human α‐thrombin complexed with BMS‐186282 and BMS‐189090
Authors: Malley Mary F. , Sack John S. , Tabernero Lydia , Chang Chiehying Y. , Ohringer Shari L. , Roberts Daniel G. M. , Das Jagabandhu 
Published: July 2010

AbstractThe crystallographic structures of the ternary complexes of human α‐thrombin with hirugen (a sulfated hirudin fragment) and the small‐molecule active site thrombin inhibitors BMS‐186282 and BMS‐189090 have been determined at 2.6 and 2.8 Å. In both cases, the inhibitors, which adopt very‐similar bound conformations, bind in an antiparallel β‐strand arrangement relative to the thrombin main chain in a manner like that reported for PPACK, D‐Phe‐Pro‐Arg‐CH2Cl. They do, however, exhibit differences in the binding of the alkyl guanidine moiety in the specificity pocket. Numerous hydrophilic and hydrophobic interactions serve to stabilize the inhibitors in the binding pocket. Although PPACK forms covalent bonds to both serine and the histidine of the catalytic triad of thrombin, neither BMS‐186282 nor BMS‐189090 bind covalently and only BMS‐186282 forms a hydrogen bond to the serine of the catalytic triad. Both inhibitors bind with high affinity (Ki = 79 nM and 3.6 nM, respectively) and are highly selective for thrombin over trypsin and other serine proteases.