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Author (up) Dingley, A.J.; Lorenzen, I.; Grotzinger, J. file  url
  Title NMR analysis of viral protein structures Type Journal Article
  Year 2008 Publication Methods in Molecular Biology (Clifton, N.J.) Abbreviated Journal Methods Mol Biol  
  Volume 451 Issue Pages 441-462  
  Keywords Cloning, Molecular; DNA, Complementary; Indicators and Reagents; Magnetic Resonance Spectroscopy--methods; Models, Molecular; Protein Conformation; Recombinant Proteins--chemistry; Solutions; Viral Proteins--chemistry, genetics, isolation & purification  
  Abstract Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool to study the three-dimensional structure of proteins and nucleic acids at atomic resolution. Since the NMR data can be recorded in solution, conditions such as pH, salt concentration, and temperature can be adjusted so as to closely mimic the biomacromolecules natural milieu. In addition to structure determination, NMR applications can investigate time-dependent phenomena, such as dynamic features of the biomacromolecules, reaction kinetics, molecular recognition, or protein folding. The advent of higher magnetic field strengths, new technical developments, and the use of either uniform or selective isotopic labeling techniques, currently allows NMR users the opportunity to investigate the tertiary structure of biomacromolecules of approximately 50 kDa. This chapter will outline the basic protocol for structure determination of proteins by NMR spectroscopy. In general, there are four main stages: (i) preparation of a homogeneous protein sample, (ii) the recording of the NMR data sets, (iii) assignment of the spectra to each NMR observable atom in the protein, and (iv) generation of structures using computer software and the correctly assigned NMR data.  
  Call Number Serial 782  
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Author (up) Haslbeck, M.; Franzmann, T.; Weinfurtner, D.; Buchner, J. file  url
  Title Some like it hot: the structure and function of small heat-shock proteins Type Journal Article
  Year 2005 Publication Nature Structural & Molecular Biology Abbreviated Journal Nat Struct Mol Biol  
  Volume 12 Issue 10 Pages 842-846  
  Keywords Heat-Shock Proteins, Small/*chemistry/classification/*metabolism; Phylogeny; Protein Conformation; Protein Folding  
  Abstract Small heat-shock proteins (sHsps) are a widespread and diverse class of molecular chaperones. Recent evidence suggests that they maintain protein homeostasis by binding proteins in non-native conformations, thereby preventing substrate aggregation. Some members of the sHsp family are inactive or only partially active under physiological conditions, and transition toward the active state is induced by specific triggers, such as elevated temperature. Release of substrate proteins bound to sHsps requires cooperation with ATP-dependent chaperones, suggesting that sHsps create a reservoir of non-native proteins for subsequent refolding.  
  Call Number Serial 2181  
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Author (up) Jaswal, S.S. file  url
  Title Biological insights from hydrogen exchange mass spectrometry Type Journal Article
  Year 2013 Publication Biochimica et Biophysica Acta Abbreviated Journal Biochim Biophys Acta  
  Volume 1834 Issue 6 Pages 1188-1201  
  Keywords Deuterium/chemistry; Deuterium Exchange Measurement/methods; Hydrogen/*chemistry; Kinetics; Mass Spectrometry/*methods; Protein Conformation; Protein Folding; Thermodynamics  
  Abstract Over the past two decades, hydrogen exchange mass spectrometry (HXMS) has achieved the status of a widespread and routine approach in the structural biology toolbox. The ability of hydrogen exchange to detect a range of protein dynamics coupled with the accessibility of mass spectrometry to mixtures and large complexes at low concentrations result in an unmatched tool for investigating proteins challenging to many other structural techniques. Recent advances in methodology and data analysis are helping HXMS deliver on its potential to uncover the connection between conformation, dynamics and the biological function of proteins and complexes. This review provides a brief overview of the HXMS method and focuses on four recent reports to highlight applications that monitor structure and dynamics of proteins and complexes, track protein folding, and map the thermodynamics and kinetics of protein unfolding at equilibrium. These case studies illustrate typical data, analysis and results for each application and demonstrate a range of biological systems for which the interpretation of HXMS in terms of structure and conformational parameters provides unique insights into function. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.  
  Call Number Serial 975  
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Author (up) Luger, K.; Mader, A.W.; Richmond, R.K.; Sargent, D.F.; Richmond, T.J. file  url
  Title Crystal structure of the nucleosome core particle at 2.8 A resolution Type Journal Article
  Year 1997 Publication Nature Abbreviated Journal Nature  
  Volume 389 Issue 6648 Pages 251-260  
  Keywords Amino Acid Sequence; Base Composition; Crystallography, X-Ray; DNA/*chemistry; DNA, Superhelical/chemistry; Histones/chemistry; Humans; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Nucleosomes/*chemistry; Protein Binding; Protein Conformation; Protein Folding  
  Abstract The X-ray crystal structure of the nucleosome core particle of chromatin shows in atomic detail how the histone protein octamer is assembled and how 146 base pairs of DNA are organized into a superhelix around it. Both histone/histone and histone/DNA interactions depend on the histone fold domains and additional, well ordered structure elements extending from this motif. Histone amino-terminal tails pass over and between the gyres of the DNA superhelix to contact neighbouring particles. The lack of uniformity between multiple histone/DNA-binding sites causes the DNA to deviate from ideal superhelix geometry.  
  Call Number Serial 1989  
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Author (up) Narayana, N.; Matthews, D.A.; Howell, E.E.; Nguyen-huu, X. file  url
  Title A plasmid-encoded dihydrofolate reductase from trimethoprim-resistant bacteria has a novel D2-symmetric active site Type Journal Article
  Year 1995 Publication Nature Structural Biology Abbreviated Journal Nat Struct Biol  
  Volume 2 Issue 11 Pages 1018-1025  
  Keywords Amino Acid Sequence; Binding Sites; Crystallography, X-Ray; Folic Acid Antagonists/chemistry/metabolism; Models, Molecular; Molecular Sequence Data; NADP/chemistry/metabolism; Plasmids/*genetics; Protein Conformation; Recombinant Proteins/chemistry; Tetrahydrofolate Dehydrogenase/*chemistry/genetics; Trimethoprim/chemistry/metabolism; Trimethoprim Resistance/*genetics  
  Abstract Bacteria expressing R67-plasmid encoded dihydrofolate reductase (R67 DHFR) exhibit high-level resistance to the antibiotic trimethoprim. Native R67 DHFR is a 34,000 M(r) homotetramer which exists in equilibrium with an inactive dimeric form. The structure of native R67 DHFR has now been solved at 1.7 A resolution and is unrelated to that of chromosomal DHFR. Homotetrameric R67 DHFR has an unusual pore, 25 A in length, passing through the middle of the molecule. Two folate molecules bind asymmetrically within the pore indicating that the enzyme's active site consists of symmetry related binding surfaces from all four identical units.  
  Call Number Serial 1208  
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Author (up) Pace, C.N. file  url
  Title Determination and analysis of urea and guanidine hydrochloride denaturation curves Type Journal Article
  Year 1986 Publication Methods in Enzymology Abbreviated Journal Methods Enzymol  
  Volume 131 Issue Pages 266-280  
  Keywords Guanidine; *Guanidines; Kinetics; *Protein Conformation; *Protein Denaturation; Spectrometry, Fluorescence; *Urea  
  Call Number Serial 259  
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Author (up) Pantoliano, M.W.; Valentine, J.S.; Burger, A.R.; Lippard, S.J. file  url
  Title A pH-dependent superoxide dismutase activity for zinc-free bovine erythrocuprein. Reexamination of the role of zinc in the holoprotein Type Journal Article
  Year 1982 Publication Journal of Inorganic Biochemistry Abbreviated Journal J Inorg Biochem  
  Volume 17 Issue 4 Pages 325-341  
  Keywords Amino Acid Sequence; Animals; Blood Proteins/*metabolism; Cattle; Erythrocytes/*metabolism; Kinetics; Metalloproteins/*metabolism; Oxidation-Reduction; Protein Conformation; Superoxide Dismutase/*metabolism; Zinc/*pharmacology  
  Abstract The zinc-free derivative of bovine erythrocuprein, Cu2E2BE, was prepared and its superoxide dismutase activity was measured and compared with that of the holoprotein, Cu2Zn2BE. The dismutase activity of these proteins was measured by quantitating their inhibition of the superoxide-mediated autooxidation of 6-hydroxydopamine, dihydroxyfumaric acid, pyrogallol, and epinephrine. It was found that the superoxide dismutase activity of the zinc-free protein is pH dependent, ranging between 82 +/- 5% (relative to Cu2Zn2BE) at pH 5.8, and 25 +/- 10% at pH 10.2. The overlapping range of assays and buffers verified that these measurements are independent of the method of assay, buffer, and ionic strength (in the range of mu = 0.10 to 0.20). The variation in activity with pH is probably due, at least in part, to the migration of Cu(II) at high pH as described previously [J. S. Valentine, M. W. Pantoliano, P. J. McDonnell, A. R. Burger, and S. J. Lippard, Proc. Natl. Acad. Sci. USA 76, 4245 (1979)], since Cu(II) bound at the zinc binding site has been shown to have little or no dismutase activity. The observation of high activity (82%) for the zinc-free protein at pH 5.8, where Cu(II) is predominantly in the native Cu binding site, and less susceptible to removal by ethylenediaminetetraacetic acid, demonstrates that the presence of Zn(II) in Cu2Zn2BE does not greatly enhance the inherent dismutase activity of Cu(II) in the holoprotein.  
  Call Number Serial 124  
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Author (up) Tsutsui, Y.; Wintrode, P.L. file  url
  Title Hydrogen/deuterium exchange-mass spectrometry: a powerful tool for probing protein structure, dynamics and interactions Type Journal Article
  Year 2007 Publication Current Medicinal Chemistry Abbreviated Journal Curr Med Chem  
  Volume 14 Issue 22 Pages 2344-2358  
  Keywords Deuterium; Deuterium Exchange Measurement/*methods; Hydrogen; Mass Spectrometry/*methods; Molecular Structure; Protein Binding; Protein Conformation; Proteins/*chemistry/*metabolism  
  Abstract Knowledge of the structure and dynamics of proteins and protein assemblies is critical both for understanding the molecular basis of physiological and patho-physiological processes and for guiding drug design. While X-ray crystallography and nuclear magnetic resonance spectroscopy are both excellent techniques for this purpose, both suffer from limitations, including the requirement for high quality crystals and large amounts of material. Recently, hydrogen/deuterium exchange measured using mass spectrometry (HXMS) has emerged as a powerful new tool for the study of protein structure, dynamics and interactions in solution. HXMS exploits the fact that backbone amide hydrogens can exchange with deuterium when a protein is incubated in D(2)O, and that the rate of the exchange process is highly dependent on the local structural environment. Several features of HXMS make it an especially attractive approach, including small sample requirements and the ability to study extremely large protein assemblies that are not amenable to other techniques. Here, we provide an overview of HXMS and describe several recent applications to problems of medical interest. After reviewing the molecular basis of the H/D exchange process, the different steps of the HXMS experiment--labeling, rapid proteolysis, fragment separation and mass measurement--are described, followed by a discussion of data analysis methods. Finally, we describe recent results on the application of HXMS to 1) mapping drug/inhibitor binding sites and detecting drug induced conformational changes, 2) studying viral capsid structure and assembly, and 3) characterizing the structure of pathological protein conformations, specifically amyloid fibrils.  
  Call Number Serial 974  
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Author (up) Van de Ven, W.J.; Creemers, J.W.; Roebroek, A.J. file  url
  Title Furin: the prototype mammalian subtilisin-like proprotein-processing enzyme. Endoproteolytic cleavage at paired basic residues of proproteins of the eukaryotic secretory pathway Type Journal Article
  Year 1991 Publication Enzyme Abbreviated Journal Enzyme  
  Volume 45 Issue 5-6 Pages 257-270  
  Keywords Animals; Binding Sites; Catalysis; Cloning, Molecular; Drosophila melanogaster; Furin; Humans; Invertebrate Hormones/genetics/metabolism; Mice; Models, Molecular; Multigene Family; Protein Conformation; Protein Precursors/*metabolism; *Protein Processing, Post-Translational; Sequence Homology, Amino Acid; Substrate Specificity; Subtilisins/genetics/*metabolism  
  Abstract Furin, the translational product of the recently discovered fur gene, appears to be the first known mammalian member of the subtilisin family of serine proteases and the first known mammalian proprotein-processing enzyme with cleavage selectivity for paired basic amino acid residues. Structurally and functionally, it resembles the prohormone-processing enzyme, kexin (EC, which is encoded by the KEX2 gene of yeast Saccharomyces cerevisiae. Most likely, furin is primarily involved in the processing of precursors of proteins that are secreted via the constitutive secretory pathway. Here, we review the discovery of the fur gene and describe the isolation of cDNA clones corresponding to human and mouse fur and to two fur-like genes of Drosophila melanogaster, Dfur1 and Dfur2. We also compare the structural organization of the various deduced furin proteins to that of yeast kexin, and of other members of the subtilisin family of serine proteases. Furthermore, the biosynthesis of biologically active human and mouse furin is evaluated. Finally, the cleavage specificity for paired basic amino acid residues of human and mouse furin is demonstrated by the correct processing of the precursor for von Willebrand factor.  
  Call Number Serial 524  
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Author (up) Weaver, T.; Lees, M.; Zaitsev, V.; Zaitseva, I.; Duke, E.; Lindley, P.; McSweeny, S.; Svensson, A.; Keruchenko, J.; Keruchenko, I.; Gladilin, K.; Banaszak, L. file  url
  Title Crystal structures of native and recombinant yeast fumarase Type Journal Article
  Year 1998 Publication Journal of Molecular Biology Abbreviated Journal J Mol Biol  
  Volume 280 Issue 3 Pages 431-442  
  Keywords Binding Sites; Crystallography, X-Ray; Fumarate Hydratase/*chemistry; Fungal Proteins/*chemistry; Models, Molecular; Polymers/chemistry; *Protein Conformation; Saccharomyces cerevisiae/*enzymology; Water/chemistry  
  Abstract Crystal structures for both native and recombinant forms of yeast fumarase from Saccharomyces cerevisiae have been completed to moderate resolution by two separate laboratories. The recombinant form was obtained by the construction of an expression plasmid for Escherichia coli. Despite a high level of amino acid sequence similarity, purification of the eukaryotic enzyme from the wild-type prokaryotic enzyme was feasible. The crystal structure of the native form, NY-fumarase, encompasses residues R22 through M484, while the recombinant form, RY-fumarase, consists of residues S27 through L485. Both crystal structures lack the N-terminal translocation segment. Each subunit of the homo-tetrameric protein has three domains. The active site is formed by segments from each of three polypeptide chains. The results of these studies on the eukaryotic proteins are unique, since the recombinant form was done in the absence of dicarboxylic acid and has an unoccupied active site. As a comparison, native fumarase was crystallized in the presence of the competitive inhibitor, meso-tartrate. Meso-tartrate occupies a position close to that of the bound citrate molecule found in the active site of the E. coli enzyme. This inhibitor participates in hydrogen bonding to an active-site water molecule. The independent determination of the two structures provides further evidence that an active-site water molecule may play an active role in the fumarase-catalyzed reaction.  
  Call Number Serial 1178  
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