bluuebunny/crossref_metadata_2025_split huggingface dataset:
Compactness of the kinetic molten globule of bovineα‐lactalbumin: A dynamic light scattering study
Authors: Gast Klaus , Zirwer Dietrich , MÜller‐Frohne Marlies , Damaschun Gregor 
Published: February 2009

AbstractDuring folding of globular proteins, the molten globule state was observed as an equilibrium intermediate under mildly denaturing conditions as well as a transient intermediate in kinetic refolding experiments. While the high compactness of the equilibrium intermediate ofα‐lactalbumin has been verified, direct measurements of the compactness of the kinetic intermediate have not been reported until now. Our dynamic light scattering measurements provide a complete set of the hydrodynamic dimensions of bovineα‐lactalbumin in different conformational states, particularly in the kinetic molten globule state. The Stokes radii for the native, kinetic molten globule, equilibrium molten globule, and unfolded states are 1.91, 1.99, 2.08, and 2.46 nm, respectively. Therefore, the kinetic intermediate appears to be even more compact than its equilibrium counterpart. Remarkable differences in the concentration dependence of the Stokes radius exist revealing strong attractive but repulsive intermolecular interactions in the kinetic and equilibrium molten globule states, respectively. This underlines the importance of extrapolation to zero protein concentration in measurements of the molecular compactness.
The role of quaternary interactions on the stability and activity of ascorbate peroxidase
Authors: Mandelman David , Li Huiying , Poulos Thomas L. , Schwarz Frederick P. 
Published: February 2009

AbstractPoint mutations at the dimer interface of the homodimeric enzyme ascorbate peroxidase (APx) were constructed to assess the role of quaternary interactions in the stability and activity of APx. Analysis of the APx crystal structure shows that Glu 112 forms a salt bridge with Lys20 and Arg24 of the opposing subunit near the axis of dyad symmetry between the subunits. Two point mutants, E112A and E112K, were made to determine the effects of a neutral (alanine) and repulsive (lysine) mutation on dimerization, stability, and activity. Gel filtration analysis indicated that the ratio of the monomer to dimer increased as the dimer interface interactions went from attractive to repulsive. Differential scanning calorimetry (DSC) data exhibited a decrease in both the transition temperature (Tm) and enthalpy of unfolding (ΔHc) with Tm = 58.3 ± 0.5 °C, 56.0 ± 0.8 °C, and 53.0 ± 0.9 °C and ΔHc = 245 ± 29 kcal/mol, 199 ± 38 kcal/mol, and 170 ± 25 kcal/mol for wild‐type APx, E112A, and E112K, respectively. Similar changes were observed based on thermal melting curves obtained by absorption spectroscopy. No change in enzyme activity was found for the E112A mutant, and only a 25% drop in activity was observed for the El 12K mutant which demonstrates that the non‐Michaelis Menten kinetics of APx is not due to the APx oligomeric structure. The cryogenic crystal structures of the wild‐type and mutant proteins show that mutation induced changes are limited to the dimer interface including an alteration in solvent structure.
Structural characterizations of nonpeptidic thiadiazole inhibitors of matrix metalloproteinases reveal the basis for stromelysin selectivity
Authors: Finzel B. C. , Baldwin E. T. , Bryant G. L. , Hess G. F. , Wilks J. W. , Trepod C. M. , Mott J. E. , Marshall V. P. , Petzold G. L. , Poorman R. A. , O'Sullivan T. J. , Schostarez H. J. , Mitchell M. A. 
Published: February 2009

AbstractThe binding of two 5‐substituted‐l,3,4‐thiadiazole‐2‐thione inhibitors to the matrix metalloproteinase stromelysin (MMP‐3) have been characterized by protein crystallography. Both inhibitors coordinate to the catalytic zinc cation via an exocyclic sulfur and lay in an unusual position across the unprimed (P1‐P3) side of the proteinase active site. Nitrogen atoms in the thiadiazole moiety make specific hydrogen bond interactions with enzyme structural elements that are conserved across all enzymes in the matrix metalloproteinase class. Strong hydrophobic interactions between the inhibitors and the side chain of tyrosine‐155 appear to be responsible for the very high selectivity of these inhibitors for stromelysin. In these enzyme/inhibitor complexes, the S1′ enzyme subsite is unoccupied. A conformational rearrangement of the catalytic domain occurs that reveals an inherent flexibility of the substrate binding region leading to speculation about a possible mechanism for modulation of stromelysin activity and selectivity.
Construction, expression, and characterization of a novel fully activated recombinant single‐chain hepatitis C virus protease
Authors: Taremi S. Shane , Beyer Brian , Maher Maureen , Yao Nanhua , Prosise Winifred , Weber Patricia C. , Malcolm Bruce A. 
Published: February 2009

AbstractEfficient proteolytic processing of essential junctions of the hepatitis C virus (HCV) polyprotein requires a heterodimeric complex of the NS3 bifunctional protease/helicase and the NS4A accessory protein. A single‐chain recombinant form of the protease has been constructed in which NS4A residues 21‐32 (GSVVIVGRIILS) were fused in frame to the amino terminus of the NS3 protease domain (residues 3ndash181) through a tetrapeptide linker. The single‐chain recombinant protease has been overexpressed as a soluble protein in E. coli and purified to homogeneity by a combination of metal chelate and size‐exclusion chromatography. The single‐chain recombinant protease domain shows full proteolytic activity cleaving the NS5A‐5B synthetic peptide substrate, DTEDVVCCSMSYTWTGK with a Km and kcat of 20.0 ± 2.0 μM and 9.6 ± 2.0 min−1, respectively; parameters identical to those of the authentic NS31_631/NS4A1_54 protein complex generated in eukaryotic cells (Sali DL et al., 1998, Biochemistry 37:3392‐3401).
Structural characterization of the entire 1.3S subunit of transcarboxylase from Propionibacterium shermanii
Authors: Reddy D. Venkat , Rothemund Sven , Sönnichsen Frank D. , Shenoy Bhami C. , Carey Paul R. 
Published: February 2009

AbstractTranscarboxylase (TC) from Propionibacterium shermanii, a biotin‐dependent enzyme, catalyzes the transfer of a carboxyl group from methylmalonyl‐CoA to pyruvate in two partial reactions. Within the multisubunit enzyme complex, the 1.3S subunit functions as the carboxyl group carrier. The 1.3S is a 123‐amino acid polypeptide (12.6 kDa), to which biotin is covalently attached at Lys 89. We have expressed 1.3S in Escherichia coli with uniform 15N labeling. The backbone structure and dynamics of the protein have been characterized in aqueous solution by three‐dimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy. The secondary structure elements in the protein were identified based on NOE information, secondary chemical shifts, homonuclear 3Jhnhα coupling constants, and amide proton exchange data. The protein contains a predominantly disordered N‐terminal half, while the C‐terminal half is folded into a compact domain comprising eight β‐strands connected by short loops and turns. The topology of the C‐terminal domain is consistent with the fold found in both carboxyl carrier and lipoyl domains, to which this domain has approximately 26‐30% sequence similarity.
Topological mimicry and epitope duplication in the guanylyl cyclase C receptor
Authors: Nandi Animesh , Visweswariah Sandhya S. , Suguna K. , Surolia Avadhesha 
Published: February 2009

AbstractGuanylyl cyclase C (GCC) is the receptor for the gastrointestinal hormones, guanylin, and uroguanylin, in addition to the bacterial heat‐stable enterotoxins, which are one of the major causes of watery diarrhea the world over. GCC is expressed in intestinal cells, colorectal tumor tissue and tumors originating from metastasis of the colorectal carcinoma. We have earlier generated a monoclonal antibody to human GCC, GCC:B10, which was useful for the immunohisto‐chemical localization of the receptor in the rat intestine (Nandi A et al., 1997, J Cell Biochem 66:500‐511), and identified its epitope to a 63‐amino acid stretch in the intracellular domain of GCC. In view of the potential that this antibody has for the identification of colorectal tumors, we have characterized the epitope for GCC:B10 in this study. Overlapping peptide synthesis indicated that the epitope was contained in the sequence HIPPENIFPLE. This sequence was unique to GCC, and despite a short stretch of homology with serum amyloid protein and pertussis toxin, no cross reactivity was detected. The core epitope was delineated using a random hexameric phage display library, and two categories of sequences were identified, containing either a single, or two adjacent proline residues. No sequence identified by phage display was identical to the epitope present in GCC, indicating that phage sequences represented mimotopes of the native epitope. Alignment of these sequences with HIPPENIFPLE suggested duplication of the recognition motif, which was confirmed by peptide synthesis. These studies allowed us not only to define the requirements of epitope recognition by GCC:B10 monoclonal antibody, but also to describe a novel means of epitope recognition involving topological mimicry and probable duplication of the cognate epitope in the native guanylyl cyclase C receptor sequence.
Effects of pressure on the structure of metmyoglobin: Molecular dynamics predictions for pressure unfolding through a molten globule intermediate
Authors: Floriano Wely B. , Nascimento Marco A. C. , Domont Gilberto B. , Goddard William A. 
Published: February 2009

AbstractWe investigated the pathway for pressure unfolding of metmyoglobin using molecular dynamics (MD) for a range of pressures (0. 1 MPa to 1. 2 GPa) and a temperature of 300 K. We find that the unfolding of metmyoglobin proceeds via a two‐step mechanism native → molten globule intermediate → unfolded, where the molten globule forms at 700 MPa. The simulation describes qualitatively the experimental behavior of metmyoglobin under pressure. We find that unfolding of the alpha‐helices follows the sequence of migrating hydrogen bonds (i, i + 4) → (i, i + 2).
Toward a universal inhibitor of retroviral proteases: Comparative analysis of the interactions of LP‐130 complexed with proteases from HIV‐1, FIV, and EIAV
Authors: Kervinen Jukka , Lubkowski Jacek , Zdanov Alexander , Bhatt Deepa , Dunn Ben M. , Hui Kwan Y. , Powell David J. , Kay John , Wlodawer Alexander , Gustchina Alla 
Published: February 2009

AbstractOne of the major problems encountered in antiviral therapy against AIDS is the emergence of viral variants that exhibit drug resistance. The sequences of proteases (PRs) from related retroviruses sometimes include, at structurally equivalent positions, amino acids identical to those found in drug‐resistant forms of HIV‐1 PR. The statine‐based inhibitor LP‐130 was found to be a universal, nanomolar‐range inhibitor against all tested retroviral PRs. We solved the crystal structures of LP‐130 in complex with retroviral PRs from HIV‐1, feline immunodeficiency virus, and equine infectious anemia virus and compared the structures to determine the differences in the interactions between the inhibitor and the active‐site residues of the enzymes. This comparison shows an extraordinary similarity in the binding modes of the inhibitor molecules. The only exceptions are the different conformations of naphthylalanine side chains at the P3/P3′ positions, which might be responsible for the variation in the Ki values. These findings indicate that successful inhibition of different retroviral PRs by LP‐130 is achieved because this compound can be accommodated without serious conformational differences, despite the variations in the type of residues forming the active‐site region. Although strong, specific interactions between the ligand and the enzyme might improve the potency of the inhibitor, the absence of such interactions seems to favor the universality of the compound. Hence, the ability of potential anti‐AIDS drugs to inhibit multiple retroviral PRs might indicate their likelihood of not eliciting drug resistance. These studies may also contribute to the development of a small‐animal model for preclinical testing of antiviral compounds.
The equilibrium unfolding of Azotobacter vinelandii apoflavodoxin II occurs via a relatively stable folding intermediate
Authors: Van Mierlo Carlo P. M. , Van Dongen Walter M. A. M. , Vergeldt Frank , Van Berkel Willem J. H. , Steensma Elles 
Published: February 2009

AbstractA flavodoxin from Azotobacter vinelandii is chosen as a model system to study the folding of α/ß doubly wound proteins. The guanidinium hydrochloride induced unfolding of apoflavodoxin is demonstrated to be reversible. Apoflavodoxin thus can fold in the absence of the FMN cofactor. The unfolding curves obtained for wild‐type, C69A and C69S apoflavodoxin as monitored by circular dichroism and fluorescence spectroscopy do not coincide. Apoflavodoxin unfolding occurs therefore not via a simple two‐state mechanism. The experimental data can be described by a three‐state mechanism of apoflavodoxin equilibrium unfolding in which a relatively stable intermediate is involved. The intermediate species lacks the characteristic tertiary structure of native apoflavodoxin as deduced from fluorescence spectroscopy, but has significant secondary structure as inferred from circular dichroism spectroscopy. Both spectroscopic techniques show that thermally‐induced unfolding of apoflavodoxin also proceeds through formation of a similar molten globule‐like species. Thermal unfolding of apoflavodoxin is accompanied by anomalous circular dichroism characteristics: the negative ellipticity at 222 nM increases in the transition zone of unfolding. This effect is most likely attributable to changes in tertiary interactions of aromatic side chains upon protein unfolding. From the presented results and hydrogen/deuterium exchange data, a model for the equilibrium unfolding of apoflavodoxin is presented.
pH‐Induced conformational changes of membrane‐bound influenza hemagglutinin and its effect on target lipid bilayers
Authors: Gray Cameron , Tamm Lukas K. 
Published: February 2009

AbstractInfluenza virus hemagglutinin (HA) has served as a paradigm for both pH‐dependent and ‐independent viral membrane fusion. Although large conformational changes were observed by X‐ray crystallography when soluble fragments of HA were subjected to fusion‐pH conditions, it is not clear whether the same changes occur in membrane‐bound HA, what the spatial relationship is between the conformationally changed HA and the target and viral membranes, and in what way HA perturbs the target membrane at low pH. We have taken a spectroscopic approach using an array of recently developed FTIR techniques to address these questions. Difference attenuated total reflection FTIR spectroscopy was employed to reveal reversible and irreversible components of the pH‐induced conformational change of the membrane‐bound bromelain fragment of HA, BHA. Additional proteolytic fragments of BHA were produced which permitted a tentative assignment of the observed changes to the HA1 and HA2 subunits, respectively. The membrane‐bound HA1 subunit undergoes a reversible conformational change, which most likely involves the loss of a small proportion of ß‐sheet at low pH. BHA was found to undergo a partially reversible tilting motion relative to the target membrane upon exposure to pH 5, indicating a previously undescribed hinge near the anchoring point to the target membrane. Time‐resolved amide H/D exchange experiments revealed a more dynamic (tertiary) structure of membrane‐bound BHA and its HA2, but not its HA1, subunit. Finally BHA and, to a lesser degree, HA1 perturbed the lipid bilayer of the target membrane at the interface, as assessed by spectral changes of the lipid ester carbonyl groups. These results are discussed in the context of a complementary study of HA that was bound to viral membranes through its transmembrane peptide (Gray C, Tamm LK, 1997, Protein Sci 6: 1993–2006). A distinctive role for the HA1 subunit in the conformational change of HA becomes apparent from these combined studies.
Addition of side‐chain interactions to 310‐helix/coil and α‐helix/310‐helix/coil theory
Authors: Sun Jia Ke , Doig Andrew J. 
Published: February 2009

AbstractAn increasing number of experimental and theoretical studies have demonstrated the importance of the 310‐helix/α‐helix/coil equilibrium for the structure and folding of peptides and proteins. One way to perturb this equilibrium is to introduce side‐chain interactions that stabilize or destabilize one helix. For example, an attractive i, i + 4 interaction, present only in the α‐helix, will favor the α‐helix over 310, while an i, i + 4 repulsion will favor the 310‐helix over α. To quantify the 310/α/coil equilibrium, it is essential to use a helix/coil theory that considers the stability of every possible conformation of a peptide. We have previously developed models for the 310‐helix/coil and 310‐helix/α‐helix/coil equilibria. Here we extend this work by adding i, i + 3 and i, i + 4 side‐chain interaction energies to the models. The theory is based on classifying residues into α‐helical, 310‐helical, or nonhelical (coil) conformations. Statistical weights are assigned to residues in a helical conformation with an associated helical hydrogen bond, a helical conformation with no hydrogen bond, an N‐cap position, a C‐cap position, or the reference coil conformation plus i, i + 3 and i, i + 4 side‐chain interactions. This work may provide a framework for quantitatively rationalizing experimental work on isolated 310‐helices and mixed 310‐/α‐helices and for predicting the locations and stabilities of these structures in peptides and proteins. We conclude that strong i, i + 4 side‐chain interactions favor α‐helix formation, while the 310‐helix population is maximized when weaker i, i + 4 side‐chain interactions are present.
Identification of tryptophan oxidation products in bovine α‐crystallin
Authors: Finley Eric L. , Dillon James , Crouch Rosalie K. , Schey Kevin L. 
Published: February 2009

AbstractOxidation is known to affect the structure, activity, and rate of degradation of proteins, and is believed to contribute to a variety of pathological conditions. Metal‐catalyzed oxidation (MCO) is a primary oxidizing system in many cell types. In this study, the oxidative effects of a MCO system (the Fenton reaction) on the structure of the tryptophan residues of α‐crystallin were determined. Tandem mass spectrometry (MS/MS) was utilized to identify specific tryptophan and methionine oxidation products in the bovine α‐crystallin sequence. After oxidative exposure, α‐crystallin was digested with trypsin, and the resulting peptides were fractionated by reverse‐phase HPLC. Structural analysis by mass spectrometry revealed that tryptophan 9 of αA‐ and tryptophan 60 of αB‐crystallin were each converted into hydroxytryptophans (HTRP), N‐formylkynurenine (NFK), and kynurenine (KYN). However, only HTRP and KYN formation were detected at residue 9 of αB‐crystallin. Oxidation of methionine 1 of αA‐ and methionine 1 and 68 of αB‐crystallin was also detected. The products NFK and KYN are of particular importance in the lens, as they themselves are photosensitizers that can generate reactive oxygen species (ROS) upon UV light absorption. The unambiguous identification of HTRP, NFK, and KYN in intact α‐crystallin represents the first structural proof of the formation of these products in an intact protein, and provides a basis for detailed structural analysis of oxidized proteins generated in numerous pathological conditions.
Global analysis of the thermal and chemical denaturation of the N‐terminal domain of the ribosomal protein L9 in H2O and D2O. Determination of the thermodynamic parameters, ΔH°, ΔS°, and ΔC°p, and evaluation of solvent isotope effects
Authors: Kuhlman Brian , Raleigh Daniel P. 
Published: February 2009

AbstractThe stability of the N‐terminal domain of the ribosomal protein L9, NTL9, from Baccilus stearothermophilus has been monitored by circular dichroism at various temperatures and chemical denaturant concentrations in H2O and D2O. The basic thermodynamic parameters for the unfolding reaction, ΔH°, ΔS°, and ΔC°p, were determined by global analysis of temperature and denaturant effects on stability. The data were well fit by a model that assumes stability varies linearly with denaturant concentration and that uses the Gibbs‐Helmholtz equation to model changes in stability with temperature. The results obtained from the global analysis are consistent with information obtained from individual thermal and chemical denaturations. NTL9 has a maximum stability of 3. 78±0. 25 kcal mol−1 at 14°C. ΔH°(25°C) for protein unfolding equals 9. 9±0. 7 kcal mol−1 and TΔS°(25 °C) equals 6. 2±0. 6 kcal mol−1. Cp equals 0. 53±0. 06 kcal mol−1 deg−1. There is a small increase in stability when D2O is substituted for H2O. Based on the results from global analysis, NTL9 is 1. 06±0. 60 kcal mol−1 more stable in D2O at 25 °C and Tm is increased by 5. 8±3. 6°C in D2O. Based on the results from individual denaturation experiments, NTL9 is 0. 68±0. 68 kcal mol−1 more stable in D2O at 25°C and Tm is increased by 3. 5±2. 1°C in D2O. Within experimental error there are no changes in ΔH°(25°C) when D2O is substituted for H2O.
Prediction by a neural network of outer membrane β‐strand protein topology
Authors: Diederichs Kay , Freigang Jörg , Umhau Stephan , Zeth Kornelius , Breed Jason 
Published: February 2009

AbstractAn artificial neural network (NN) was trained to predict the topology of bacterial outer membrane (OM) ß‐strand proteins. Specifically, the NN predicts the z‐coordinate of Cα atoms in a coordinate frame with the outer membrane in the xy‐plane, such that low z‐values indicate periplasmic turns, medium z‐values indicate transmembrane ß‐strands, and high z‐values indicate extracellular loops. To obtain a training set, seven OM proteins (porins) with structures known to high resolution were aligned with their pores along the z‐axis. The relationship between Cα z‐values and topology was thereby established. To predict the topology of other OM proteins, all seven porins were used for the training set. Z‐values (topologies) were predicted for two porins with hitherto unknown structure and for OM proteins not belonging to the porin family, all with insignificant sequence homology to the training set. The results of topology prediction compare favorably with experimental topology data.
Sequential assignment of 1H, 15N, 13C resonances and secondary structure of human calmodulin‐like protein determined by NMR spectroscopy
Authors: Qian Hong , Rogers Michael S. , Schleucher Jürgen , Edlund Ulf , Strehler Emanuel E. , Sethson Ingmar 
Published: February 2009

AbstractHuman calmodulin‐like protein (CLP) is closely related to vertebrate calmodulin, yet its unique cell specific expression pattern, overlapping but divergent biochemical properties, and specific target proteins suggest that it is not an isoform of calmodulin. To gain insight into the structural differences that may underlie the difference target specificities and biochemical properties of CLP when compared to calmodulin, we determined the sequential backbone assignment and associated secondary structure of 144 out of the 148 residues of Ca2+‐CLP by using multinuclear multidimensional NMR spectroscopy. Despite a very high overall degree of structural similarity between CLP and calmodulin, a number of significant differences were found mainly in the length of α‐helices and in the central nonhelical flexible region. Interestingly, the regions of greatest primary sequence divergence between CLP and calmodulin in helices III and VIII displayed only minor secondary structure differences. The data suggest that the distinct differences in target specificity and biochemical properties of CLP and calmodulin result from the sum of several minor structural and side‐chain changes spread over multiple domains in these proteins.