Vai ai contenuti. | Spostati sulla navigazione | Spostati sulla ricerca | Vai al menu | Contatti | Accessibilità

| Crea un account

FRASSON, ROBERTA (2009) Protein Engineering by Chemical and Genetic Methods
Applications to Protein Recognition and Thrombin Function.
[Tesi di dottorato]

Full text disponibile come:

Documento PDF

Abstract (inglese)

In the past three decades, the advent of recombinant DNA technology allowed the site-specific alteration of a given polypeptide chain at a glance, thus much expanding the tools available to study the molecular mechanisms of protein folding, stability, and function. This approach, also known as protein engineering, is based on the possibility of modifying the chemical composition of a protein at a single or multiple sites with the 20 available coded amino acids, thus obtaining recombinant mutant proteins with altered structure, function, or stability properties. Evaluation of the effects of the mutation on the property under investigation (e.g., folding, stability or molecular recognition) will allow quantitative estimation of the contribution of that specific amino acid which has been mutated in the wild-type protein. This information will in turn help in understanding the physico-chemical determinants exploited by natural polypeptide chains to spontaneously acquire a unique, stable and functionally active conformation. A comprehensive view of these fundamental processes at the molecular level is of paramount importance in the successful design of novel proteins, which is the ultimate goal of protein engineering. More recently, the incorporation of noncoded amino acids, possessing unique physico-chemical or spectroscopic properties, into proteins has emerged as a novel and promising approach in protein science. Protein engineering with non-coded amino acids, in fact, allow investigators to finely tune the structure at a protein site, thus much expanding the scope of physical-organic chemistry in the study of proteins. With respect to this, stepwise solid-phase chemical synthesis remains the easiest and fastest approach to site-specifically incorporate in high yields noncoded amino acid into even long (50-80 amino acids) polypeptide chains, approaching the size of real proteins. In this doctoral Thesis, relevant applications of protein engineering experiments by both genetic and chemical methods will be presented.
During our studies aimed to dissect the structure-function relationships of human ?-thrombin, we approached the task of devising an efficient system to produce large amounts of recombinant protein, either in native or mutated form. Thrombin is a serine protease that plays a pivotal role in haemostasis. Human thrombin is a glycoprotein consisting of two polypeptides, a 259-residue B-chain and a smaller 36-residue A-chain, connected by a disulfide bond. B-chain contains the catalytic triad of thrombin and three disulfide bonds. Hence, prethrombin-2, the smallest physiological single-chain precursor of ?-thrombin, was expressed in E. coli, a prokaryotic system which is easy to work with, to scale up, and less time-consuming than eukaryotic systems. Using this expression system, we addressed several issues in structure-function studies on thrombin. Firstly, taking advantage of the fact that E. coli lacks the biochemical machinery for conjugating carbohydrate chains to proteins, we have studied the role of glycosylation on the structure, stability and function of the wild-type recombinant protein. Secondly, with the aim to understand the effect of natural mutations (i.e., desLys9a and Gly25Ser) in the thrombin A-chain on the structure and function of the enzyme, we have produced the corresponding recombinant forms of the naturally occurring variants of thrombin. Thirdly, we produced two mutants of human thrombin in which key Arg-residues (i.e., Arg73 in exosite I and Arg101 in exosite II) in the positively charged exosite I and II binding sites were replaced by Ala, in order to abrogate ligand-binding at each exosite. In all cases, the recombinant proteins accumulated in the inclusion bodies, from which disulfide-coupled renaturation was achieved in significantly high yields in almost all cases, yielding about 10 mg per liter of fully active wild-type human ?-thrombin. All mutant proteins were subjected to thorough characterization with respect to their chemical identity, as well as conformational, stability and functional properties. A major finding of our work was that the recombinant wild-type enzyme, lacking the carbohydrate moiety, has conformational and functional properties indistinguishable from those pertaining to the natural thrombin, but it is significantly less stable than the natural counterpart.
A major application of protein engineering with noncoded amino acids entails the incorporation of suitable spectroscopic probes for studying ligand-protein and protein-protein interactions. With respect to this, 3-nitrotyrosine (NT) in absorbs radiation in the wavelength range where Tyr and Trp emit fluorescence (300-450 nm) and it is essentially nonfluorescent. Therefore, NT may function as an energy acceptor in resonance energy transfer (FRET) studies for investigating ligand-protein interactions. Here, the potentialities of NT were tested on the hirudin-thrombin system, a well-characterized protease-inhibitor pair of key pharmacological importance. We synthesized two analogues of the N-terminal domain (residues 1-47) of hirudin: Y3NT, in which Tyr3 was replaced by NT, and S2R/Y3NT, containing the substitutions Ser2?Arg and Tyr3?NT. The binding of these analogues to thrombin was investigated at pH 8 by FRET and UV/Vis-absorption spectroscopy. Upon hirudin binding, the fluorescence of thrombin was reduced by ?50%, due to the energy transfer occurring between the Trp-residues of the enzyme (i.e, the donors) and the single NT of the inhibitor (i.e., the acceptor). Our results indicate that the incorporation of NT can be effectively used to detect protein-protein interactions with sensitivity in the low nanomolar range, to uncover subtle structural features at ligand-protein interface, and to obtain reliable Kd values for structure-activity relationships studies.
High throughput screening of protein-protein and protein-peptide interactions is of high interest both for biotechnological and pharmacological applications. Here, we propose the use of the non-coded amino acids o-nitrotyrosine and p-iodophenylalanine as spectroscopic probes in combination with circular dichroism and fluorescence quenching techniques (i.e., collisional quenching and resonance energy trasfer) as a mean to determine the peptide orientation in protein-peptide complexes. The method was successfully tested on an SH3 domain from a yeast myosin which is known to recognize specifically class-I peptides. The chemical strategies outlined here highlights the broad applicability of noncoded amino acids in biotechnology and pharmacological screening.

Statistiche Download - Aggiungi a RefWorks
Tipo di EPrint:Tesi di dottorato
Dottorato (corsi e scuole):Ciclo 21 > Scuole per il 21simo ciclo > SCIENZE MOLECOLARI > SCIENZE FARMACEUTICHE
Data di deposito della tesi:13 Gennaio 2009
Anno di Pubblicazione:31 Gennaio 2009
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/10 Biochimica
Struttura di riferimento:Dipartimenti > Dipartimento di Scienze Farmaceutiche
Codice ID:1326
Depositato il:13 Gen 2009
Simple Metadata
Full Metadata
EndNote Format


I riferimenti della bibliografia possono essere cercati con Cerca la citazione di AIRE, copiando il titolo dell'articolo (o del libro) e la rivista (se presente) nei campi appositi di "Cerca la Citazione di AIRE".
Le url contenute in alcuni riferimenti sono raggiungibili cliccando sul link alla fine della citazione (Vai!) e tramite Google (Ricerca con Google). Il risultato dipende dalla formattazione della citazione.

Abraham, M.H., Du, C.M., and Platts, J.A. (2000) Lipophilicity of the nitrophenols. J. Org. Chem. 65, 7114-7118. Cerca con Google

Akhavan, S, Mannucci, P.M., Lak, M,. Mancuso, G., Mazzucconi, M.G., Rocino, A., Jenkins, P.V., and Perkins, S.J. (2000) Identification and three-dimensional structural analysis of nine novel mutations in patients with prothrombin deficiency. Thromb Haemost 84, 989–97. Cerca con Google

Akhavan, S., Miteva, M.A., Villoutreix, B.O., Venisse, L., Peyvandi, F., Mannucci, P..M., Guillin, M.C., and Bezeaud, A. (2005) A critical role for Gly25 in the B chain of human thrombin. J. Thromb Haemost. 3, 139–145. Cerca con Google

Albericio, F. (2004) Developments in peptides and amide synthesis. Curr. Opin. Chem. Biol. 8, 211-221. Cerca con Google

Anderson, B.L., Boldogh, I., Evangelista, M., Boone, C., Greene, L.A., and Pon, L.A. (1998) The Src homology domain 3 (SH3) of a yeast type I myosin, Myo5p, binds to verprolin and is required for targeting to sites of actin polarization. J. Cell. Biol. 141, 1357-1370. Cerca con Google

Apweiler, R., Hermjakob, H., and Sharon, N. (1999) On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochim. Biophys. Acta. 1473, 4-8. Cerca con Google

Arni, R.K., Padmanabhan, K., Padmanabhan, K.P., Wu, T.P., and Tulinsky, A. (1993) Structures of the noncovalent complexes of human and bovine prothrombin fragment 2 with human PPACK-thrombin. Biochemistry. 32, 4727-4737. Cerca con Google

Ayala, Y., and Di Cera, E. (1994) Molecular Recognition by thrombin. Role of the slow ? fast transition, site-specific ion binding energetics and thermodynamic mapping of structural components. J. Mol. Biol. 235, 733-746. Cerca con Google

Ayala, Y.M., Cantwell, A.M., Rose, T., Bush, L.A., Arosio, D., and Di Cera, E. (2001) Molecular mapping of thrombin–receptor interactions. Proteins 45, 107–116. Cerca con Google

Barlos, K., Chatzi, O., Gatos, D., and Stavropoulos, G. (1991) 2-Chlorotrityl chloride resin. Studies on anchoring of Fmoc-amino acids and peptide cleavage. Int. J. Pept. Protein Res. 37, 513-520. Cerca con Google

Bell., R., Stevens, W.K., Jia, Z., Samis, J., Coté, H.C.F., MacGillivray, R.T.A., and Nesheim, M.E. (2000) Fluorescence properties and functional roles of tryptophan residues 60d, 96, 148, and 215 of thrombin. J. Biol. Chem. 275, 29513-29520. Cerca con Google

Berlman, I.B. (1973) Empirical study of heavy-atom collisional quenching of the fluorescence state of aromatic compounds in solution. J. Phys. Chem. 77, 562-567. Cerca con Google

Bode W. (2006) The structure of thrombin: A janus-headed proteinase. Semin. Thromb. Haemost. 32, 16-31. Cerca con Google

Bode, W. Maryr, I., and Baumann, U., (1989) The refined 1.9 Å crystal structure of human a-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr Pro Pro Trp insertion segment. EMBO J. 8, 3467-3475. Cerca con Google

Bode, W., Turk, D., and Karshikov, A. (1992) The refined 1.9-Å X-ray crystal structure of D-Phe-Pro-Arg-chloromethylketone-inhibited human ?-thrombin: Structure analysis, overall structure, electrostatic properties, detailed active-site geometry, and structure-function relationships. Protein Sci. 1, 426-471. Cerca con Google

Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. Cerca con Google

Brahms, S., and Brahms, J. (1980) Determination of protein secondary structure in solution by vacuum ultraviolet circular dichroism. J. Mol. Biol. 138, 149-178. Cerca con Google

Butkowski, R.J., Elio, J., Downing, M.R., and Mann, K.G. (1977) Primary structure of human prothrombin-2 and alpha-thrombin. J. Biol. Chem. 252, 4942-4957. Cerca con Google

Carpino, L.A. (1993) 1-Hydroxy-7-azabenzotriazole. An efficient peptide coupling additive. J. Am. Chem. Soc. 115, 4397-4398. Cerca con Google

Carter, W.J., Cama, E., Huntington, J.A. (2005) Crystal structure of thrombin bound to heparin. J. Biol. Chem. 280, 2745-2749. Cerca con Google

Cesareni, G., Panni, S., Nardelli, G., and Castagnoli, L. (2002) Can we infer peptide recognition specificity mediated by SH3 domains? FEBS Lett. 513, 38-44. Cerca con Google

Cestra, G., Castagnoli, L., Dente, L., Minenkova, O., Petrelli, A., Mingine, N., Hoffmuller, U., Schneider-Mergener, J., and Cesareni, G. (1999) The SH3 domain of endophilin and amphiphysin bind to the proline rich region of synaptojanin at distinct sites that display an unconventional binding specificity. J. Biol. Chem. 274, 32001-32007. Cerca con Google

Cleland JL, Craik CS. (1996). Protein Engineering. Principles and Practice. Wiley-Liss, New York. Cerca con Google

Cohen, B.E., McAnaney, T.B., Park, E.S., Jan, Y.N., Boxer, S.G., and Jan, L.Y. (2002) Probing protein electrostatics with a synthetic fluorescent amino acid. Science 296, 1700-1703. Cerca con Google

Colwell, N.S., Blinder, M.A., Tsiang, M., Gibbs, C.S., Bock, P.E., Tollefsen D.M. (1998) Allosteric effects of a monoclonal antibody against thrombin exosite II. Biochemistry. 37, 15057-65. Cerca con Google

Copeland, R.A. (2000). Enzymes: a Practical Introduction to Structure, Mechanism, and Data Analysis. 2nd ed., J. Wiley & Sons, New York. Cerca con Google

Corbett, R.J., and Roche, R.S. (1984) Use of high-speed size-exclusion chromatography for the study of protein folding and stability. Biochem. 23, 7299-7307. Cerca con Google

Cornish, V.W., Benson, D.R., Altenbach, C.A., Hideg, K., Hubbell, W.L., and Schultz, P.G. (1994) Site-specific incorporation of biophysical probes into proteins. Proc. Natl. Acad. Sci. U.S.A. 91, 2910-2924. Cerca con Google

Craik, C.S., Roczniak, S., Largman, C., Rutter, W.J. (1987). The catalytic role of the active site aspartic acid in serine protease. Science. 237, 909- 913. Cerca con Google

Cropp, A.T., and Schultz, P.G. (2004) An expanding genetic code. Trends Genet. 20, 625-630. Cerca con Google

Csizmadia, F., Tsantili-Kkoulidou, A., Panderi, I., and Darvas, F. (1997) Prediction of distribution coefficient from structure. 1. Estimation method. J. Pharm. Sci. 7, 865-871. Cerca con Google

d’Audigier, C., Pasmant, E., Bournier, O., Laurian, Y., Guillin., MC., and Bezeaud. A. (2008) A natural variant with a point mutation resulting in a homozygous Arg to His substitution at position 388 in prothrombin. Haematologica 93, 799-800. Cerca con Google

Dalgarno, D.C., Botfield, M.C., and Rickles, R.J. (1997) SH3 domains and drug design: ligands, structure, and biological function. Biopolymers 43, 383-400. Cerca con Google

Dang, Q.D., Vindigni, A., and Di Cera, E. (1995) An allosteric switch controls the procoagulant and anticoagulant activities of thrombin. Proc. Natl. Acad. Sci. USA 92, 5977-5981. Cerca con Google

Davie, E. W., Fujikawa, K., and Kisiel, W. 1991. The coagulation cascade: initiation, maintenance, and regulation. Biochemistry 30, 10363-10370. Cerca con Google

Davie, E.W., and Kulman, J.D. (2006) An overview of the structure and function of thrombin. Sem. Thromb. Haemost. 32, Suppl. 1, 3-15. Cerca con Google

Dawson, P., and Kent, S.B. (2000) Synthesis of native proteins by chemical ligation. Annu. Rev. Biochem. 69, 923-960. Cerca con Google

De Candia, E., De Cristofaro, R., De Marco, L., Mazzucato, M., Picozzi, M., and Landolfi, R. (1997) Thrombin interaction with platelet GpIB: role of the heparin binding domain. Thromb Haemost. 77, 735-740. Cerca con Google

De Candia, E., Hall, S.W., Rutella, S., Landolfi, R., Andrews, R.K., and De Cristofaro, R. (2001) Binding of thrombin to glycoprotein Ib accelerates the hydrolysis of PAR-1 on intact platelets. J. Biol. Chem. 276, 4692-4698. Cerca con Google

De Cristofaro, R., Akhavan, S., Altomare, C., Carotti, A., Peyvandi, F., and Mannucci, P.M. (2004) A natural prothrombin mutant reveals an unexpected influence of A-chain structure on the activity of human alpha-thrombin. J. Biol Chem. 279, 13035-13043. Cerca con Google

De Cristofaro, R., Akhavan, S., Altomare, C., Carotti, A., Peyvandi, F., and Mannucci, P.M. (2004) A natural prothrombin mutant reveals an unexpected influence of A-chain structure on the activity of human alpha-thrombin. J. Biol Chem. 279, 13035-13043. Cerca con Google

De Cristofaro, R., and Di Cera, E. (1990) Effect of protons on the amidase activity of human alpha-thrombin. Analysis in terms of a general linkage scheme. J. Mol. Biol. 216, 1077-1085. Cerca con Google

De Cristofaro, R., De Candia, E., Landolfi, R., Rutella, S., and Scott W. Hall, S.W. (2001) Structural and functional mapping of the thrombin domain involved in the binding to the platelet glycoprotein Ib. Biochemistry. 40, 13268-13273. Cerca con Google

De Filippis, V., Colombo, G., Russo, I., Spadari, B., and Fontana, A. (2002) Probing hirudin-thrombin interaction by incorporation of noncoded amino acids and molecular dynamics simulation. Biochemistry. 43, 1537-1550. Cerca con Google

De Filippis, V., De Boni, S., De Dea, E., Dalzoppo, D., Grandi, C., and Fontana, A. (2004) Incorporation of the fluorescent amino acid 7-azatryptophan into the core domain 1-47 of hirudin as a probe of hirudin folding and thrombin recognition. Protein Sci. 13, 1489-1502. Cerca con Google

De Filippis, V., De Dea, E., Lucatello, F., and Frasson, R. (2005) Effect of Na+ binding on the conformation, stability, and molecular recognition properties of thrombin. Biochem. J. 390, 485-492. Cerca con Google

De Filippis, V., Frasson, R., and Fontana, A. (2006) 3-Nitrotyrosine as a Spectroscopic Probe for Investigating Protein-Protein Interactions. Protein Sci. 15, 976-986. Cerca con Google

De Filippis, V., Quarzago, D., Vindigni, A., Di Cera, E., and Fontana, A. (1998) Synthesis and characterization of more potent analogues of hirudin fragment 1-47 containing non-natural amino acids. Biochemistry. 37, 13507-13515. Cerca con Google

De Filippis, V., Russo, I., Vindigni, A., Di Cera, E., Salmaso, S., and Fontana, A. (1999) Incorporation of noncoded amino acids into the N-terminal domain 1-47 of hirudin yields a highly potent and selective thrombin inhibitor. Protein Sci. 8, 2213-2217. Cerca con Google

De Filippis, V., Vindigni, A., Altichieri, L., and Fontana A. (1995) Core domain of hirudin from leech. Hirudinaria manillensis. Chemical synthesis, purification and characterization of a Trp3-analogue of fragment 1-47. Biochemistry. 34, 9552-9564. Cerca con Google

Degen, S.J., and Davie, E.W. (1987) Nucleotide sequence of the gene for human prothrombin. Biochemistry. 26, 6165-6177. Cerca con Google

Di Bella, E.E., Maurer, M.C., and Scheraga, H.A. (1995) Expression and folding of recombinant bovine prethrombin-2 and its activation to thrombin. J. Biol. Chem. 270, 163-169. Cerca con Google

Di Cera, E., Dang, Q.D., and Ayala, Y.M. (1997) Molecular mechanisms of thrombin function. Cell Mol. Life Sci. 53, 701-730. Cerca con Google

Di Nardo, A.A., Larson, S.M., and Davidson, A.R. (2003) The relationship between conservation, thermodynamic stability, and function in the SH3 domain hydrophobic core. J. Mol. Biol. 333, 641-655. Cerca con Google

Di Stasio, E., Bizzarri, P., Misiti, F., Pavoni, E., and Brancaccio, A. (2004) A fast, accurate procedure to collect and analyse unfolding fluorescence signal: the case of dystroglycan. Biophys. Chem. 107, 197-211. Cerca con Google

Dodt, J., Seemüller, U., Maschler, R., and Fritz, H. (1985) The complete covalent structure of hirudin. Localization of the disulfide bonds. Biol Chem. 366, 379-385. Cerca con Google

Dominguez, C., Boelens, R., and Bonvin, A.M. (2003) HADDOCK: A protein–protein docking approach based on biochemical or biophysical information. J. Am. Chem. Soc. 125, 1731-1737. Cerca con Google

Dos Remedios, C.G., and Moens, P.D.J. (1995) Fluorescence resonance energy transfer spectroscopy is a reliable “ruler” for measuring structural changes in proteins. Dispelling the problem of the unknown orientation factor. J. Struct. Biol. 115, 175-185. Cerca con Google

Dougherty, D.A. (2000) Un natural amino acids as probes of protein structure and function. Curr. Opin. Chem. Biol. 4, 645-652. Cerca con Google

Downing, M.R., Butkowski, R.J., Clark, M.M., and Mann, K.G. (1975) Human prothrombin activation. J. Biol. Chem. 250, 8897-8906. Cerca con Google

Dreeskamp, H., Koch, E., and Zander, M. (1975) On the fluorescence quenching of polycyclic aromatic hydrocarbons by nitromethane. Z. Naturforsch. 30a, 1311-1314. Cerca con Google

Eftink, M.R. (1997) Fluorescence methods for studying equilibrium macromolecule-ligand interactions. Methods Enzymol. 278, 221-257. Cerca con Google

Eftink, M.R. and Ghiron, C. (1981) Fluorescence quenching studies with proteins. Anal. Biochem. 114, 199-227. Cerca con Google

England, P.M. (2004) Un natural amino acid mutagenesis: a precise tool for probing protein structure and function. Biochemistry 43, 11623-11629. Cerca con Google

Esmon, C.T. (1989) The roles of protein C and thrombomodulin in the regulation in the regulation of blood coagulation. J. Biol. Chem. 264, 4743-4746. Cerca con Google

Esmon, C.T., and Jackson, C.M. (1974) The conversion of prothrombin to thrombin. III. The factor Xa-catalyzed activation of prothrombin. J. Biol. Chem. 249, 7782-7790. Cerca con Google

Esmon, C.T., and Lollar, P. (1996) Involvement of thrombin anionbinding exosites 1 and 2 in the activation of factor V and factor VIII. J Biol Chem 271, 13882–13887. Cerca con Google

Fenton, J.W. II, Fasco, M., and Stackrow, A.B. (1977) Human thrombins: production, evaluation, and properties of ?-thrombin. J. Biol. Chem. 252, 3587-3598. Cerca con Google

Fernandez-Ballester, G., Blanes-Mira, C., and Serrano, L. (2004) The tryptophan switch: changing ligand-binding specificity from type-I to type-II in SH3 domains. J. Mol. Biol. 335, 619-629. Cerca con Google

Fersht, A., and Winter, G. (1992) Protein engineering. Trends Biochem. Sci. 17, 292-295. Cerca con Google

Folkers, P.J.M., Clore, G.M., Driscoll, P.C., Dodt, J., Kohler, S., and Gronenborn, A.M. (1989) Solution structure of recombinant hirudin and the Lys-47?Glu mutant: A nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing study. Biochemistry 28, 2601-2617. Cerca con Google

Freedman, R.B. (1971) Applications of the chemical reactions of proteins in studies of their structure and function. Q. Rev. Chem. Soc. 25, 431-462. Cerca con Google

Fuentes-Prior, P. (2000) Crystal structure of the human alpha-thrombin-haemadin complex: an exosite II-binding inhibitor. EMBO J. 19, 5650-5660. Cerca con Google

Gan, Z.R., Lewis, S.D., Stone, J.R., and Shafer, J.A. (1991) Reconstitution of catalytically competent human ?-thrombin by combination of ?-thrombin residues A1-36 and B1-148 and an Escherichia coli expressed polypeptide corresponding to ?-thrombin residues B149-259. Biochemistry. 30, 11694-11699. Cerca con Google

Gan, Z.R., Li., Y., Chen, Z., Lewis, S.D., and Shafer, J.A. (1994) Identification of basic amino acid residues in thrombin essential for heparin-catalyzed inactivation by antithrombin III. J. Biol. Chem. 269, 1301-1305. Cerca con Google

Gill, S.G. and von Hippel, P.H. (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182, 319-326. Cerca con Google

Greenspan, N.S. and Di Cera, E. (1999) Defining epitopes: It’s not as easy as it seems. Nat. Biotechnol. 17, 936-937. Cerca con Google

Hageman, T.C., Endres, G.F., and Scheraga, H.A. (1975) Mechanism of action of thrombin on fibrinogen. On the role of the A chain of bovine thrombin in specificity and in differentiating between thrombin and trypsin. Arch. Biochem. Biophys. 17, 1327-1336. Cerca con Google

Hall, S.W., Nagashima, M., Zhao, L., Morser, J., and Leung, L.K. (1999) Thrombin interacts with thrombomodulin, protein C, and thrombin-activable fibrinolysis inhibitor via specific and distinct domains. J. Biol. Chem. 274, 25510-25516. Cerca con Google

Halliwell, B. (1997) What nitrates tyrosine? Is nitrotyrosine specific as a biomarker of peroxynitrite formation in vivo? FEBS Lett. 411, 157-160. Cerca con Google

Haruyama. H, and Wüthrich, K. (1989) Conformation of recombinant desulfatohirudin in aqueous solution determined by nuclear magnetic resonance. Biochemistry. 28, 4301-4312. Cerca con Google

Hendrickson, T.L., de Crécy-Lagard, V., and Schimmel, P. (2004) Incorporation of nonnatural amino acids into proteins. Annu. Rev. Biochem. 73, 147-176. Cerca con Google

Hofsteenge, J, Braun, P.J., and Stone, S.R. (1988) Enzymatic properties of proteolytic derivatives of human ?-thrombin. Biochemistry 27, 2144–2151. Cerca con Google

Hogan, J.G. Jr. (1997) Combinatorial chemistry in drug discovery. Nature Biotech. 15, 328-330. Cerca con Google

Horne, M.K., and Gralnick, H.R. (1983) The oligosaccharide of human thrombin: investigations of functional significance. Blood 1, 188-194. Cerca con Google

Hovius, R., Vallotton, P., Wohland, T., and Vogel, H. (2000) Fluorescence techniques: shedding light on ligand-receptor interactions. Trends Pharmacol. Sci. 21, 266-273. Cerca con Google

Hutchinson III, C.A., Phillips, S., Edgell, M.H., Gillam, S., Jahnke, P., ans Smith, M. (1978) Mutagenesis at a specific position in a DNA sequence. J. Biol. Chem. 253, 6551-6560. Cerca con Google

Huntington, J.A. (2008) How Na+ activates thrombin – A review of the functional and structural data. Biol. Chem. 389, 1025-1035. Cerca con Google

Huntington, J.A., and Esmon, C. (2003) The molecular basis of thrombin allostery revealed by a 1.8-Å structure of the “slow” form. Structure 11, 469-479. Cerca con Google

James, H.L., Kim, D.J., Zheng, D.Q., and Girolami, A. (1995) Prothrombin Molise I: documentation of a second incidence of replacement of a critical Arg near the active site. Thromb Res. 80, 363-366. Cerca con Google

Jameson, D.M., Croney, J.C., and Moens, P.D. (2003) Fluorescence: basic concepts, practical aspects, and some anecdotes. Methods Enzymol. 360, 1-43. Cerca con Google

Jia, C.Y.H., Nie, J., Wu, C., Li, C., and Li, S.S. (2005) Novel Src homology 3 domain-binding motifs identified from proteomic screen of a Pro-rich region. Mol. Cell. Proteomics 4, 1155-1166. Cerca con Google

Johnson, D.J.D., Adams, T.E., Li, W., and Huntington, J.A. (2005) Crystal structure of wild-type human thrombin in the Na+-free state. Biochem. J. 392, 21-28. Cerca con Google

Jones, S., and Thornton, J.M. (1996) Principles of protein-protein interactions. Proc. Natl. Acad. Sci. U.S.A. 93, 13-20. Cerca con Google

Juminaga, D., Albaugh, S.A., and Steiner, R.F. (1994) The interaction of calmodulin with regulatory peptides of phosphorylase kinase. J. Biol. Chem. 269, 1660-1667. Cerca con Google

Kang, H., Freund, C., Duke-Cohan, J.S., Musacchio, A., Wagner, G., and Rudd, C.E. (2000) SH3 domain recognition of a proline-independent tyrosine-based RkxxYxxY motif in immune cell adaptor SKAP55. EMBO J. 19, 2889-2899. Cerca con Google

Karshikov, A., Bode, W., Tulinsky, A., and Stone, S.R. (1992) Electrostatic interactions in the association of proteins: An analysis of the thrombin-hirudin complex. Protein Sci. 1, 727-735. Cerca con Google

Kay, B., Williamson, M.P., and Sudol, M. (2000) The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains. FASEB J. 14, 231-241. Cerca con Google

Kent, S.B. (1988) Chemical synthesis of peptides and proteins. Annu. Rev. Biochem. 57, 957-989. Cerca con Google

Krem, M.M., and Di Cera, E. (2003) Dissecting substrate recognition by thrombin using the inactive mutant S195A. Biophys Chem. 100, 315-323. Cerca con Google

Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Cerca con Google

Lakowicz, J.R. (1999) Principles of Fluorescence Spectroscopy 2nd ed., Kluwer Academic/Plenum, New York. Cerca con Google

Lancellotti, S., Rutella, S., De Filippis, V., Pozzi, N., and Rocca, B. (2008) Fibrinogen-elongated ? chain inhibits thrombin-induced platelet response, hindering the interaction with different receptors. J. Biol. Chem. 283, 30193-30204. Cerca con Google

Larson, S.M., and Davidson, A.R. (2000) The identification of conserved interactions within the SH3 domain by alignment of sequences and structures. Protein Sci. 9, 2170-2180. Cerca con Google

Le Bonniec, B.F., and Esmon, C.T. (1991) Glu-192 ? Gln substitution in thrombin mimics the catalytic switch induced by thrombomodulin. Proc. Natl. Acad. Sci. 88, 7371-7375. Cerca con Google

Li, S.S. (2005) Specificity and versatility of SH3 and other proline-recognition domains: structural basis and implications for cellular signal transduction. Biochem. J. 390, 641-653. Cerca con Google

Liu, L.W., Rezaie, A.R., Carson, C.W., Esmon. N.L., and Esmon, C.T. (1994) Occupancy of anion binding exosite 2 on thrombin determines Ca2+ dependence of protein C activation. J. Biol. Chem. 269, 11807-11812. Cerca con Google

Liu, L.W., Vu, T.K., Esmon, C.T., and Coughlin, S.R. (1991) The region of thrombin receptor resembling hirudin binds to thrombin and alters enzyme specificity. J. Biol. Chem. 266, 16977–16980. Cerca con Google

Lo Conte, L., Chothia, C., and Janin, J. (1999) The atomic structure of protein-protein recognition sites. J. Mol. Biol. 285, 2177-2198. Cerca con Google

Lottenberg, R., and Jackson, C.M. (1983) Solution composition dependent variation in extinction coefficients for p-nitroaniline. Biochim. Biophys. Acta. 742, 558–564. Cerca con Google

MacArthur, M.W., and Thronton, J.M. (1991) Influence of proline residues on protein conformation. J. Mol. Biol. 218, 397-412. Cerca con Google

Marcus, Y. (1994) A simple empirical model describing the thermodynamics of hydration of ions widely varying charges, sizes, and shapes. Biophys. Chem. 51, 111-127. Cerca con Google

Markwardt, F., (1994) The development of hirudin as an antithrombotic drug. Thromb. Res. 74, 1-23. Cerca con Google

Mathews, I.I., Padmanabhan, K.P., Ganesh, V., Tulinsky, A., Ishii, M., Chen, J., Turck, C.W., Coughlin, S.R., Fenton, J.W. and. (1994) Crystallographic structures of thrombin complexed with thrombin receptor peptides: existence of expected and novel binding modes. Biochemistry. 33, 3266-3279. Cerca con Google

Mattos, C. (2002) Protein-water interactions in a dynamic world. Trends Biochem. Sci. 27, 203-208. Cerca con Google

Mayer, B.J. (2001) SH3 domains: complexity in moderation. J. Cell. Sci. 114, 1253-1263. Cerca con Google

Meloun, B., Fri?, I., and Šorm, F. (1968) Nitration of tyrosine residues in the pancreatic trypsin inhibitor with tetranitromethane. Eur. J. Biochem. 4, 112-117. Cerca con Google

Mezo, A.R., Cheng, R.P., and Imperiali, B. (2001) Oligomerization of uniquely folded mini-protein motifs: development of a homotrimeric ??? peptide. J. Am. Chem. Soc. 123, 3885-3891. Cerca con Google

Mitra, N., Sinha, S., Ramya, T.N.C., and Surolia, A. (2006) N-linked oligosaccharides as outfitters for glycoprotein folding, form and functions. Trends Biochem. Sci. 31, 156-163. Cerca con Google

Mongiovì, A.M., Romano, P.R., Panni, S., Mendoza, M., Wong, W.T., Musacchio, A., Cesareni, G., and Di Fiore, P.P. (1999) A novel peptide-SH3 interaction. EMBO J. 18. 5300-5309. Cerca con Google

Morita, T., and Iwanaga, S. (1981) Prothrombin activator from Echis carinatus venom. Methods Enzymol. 80, 303-311. Cerca con Google

Morton, C.J. and Campbell, I.D. (1994) SH3 domains: molecular 'velcro'. Curr. Biol. 4, 615-617. Cerca con Google

Mosesson, M.W. (2005) Fibrinogen and fibrin structure and functions. J. Thromb. Haemost. 3, 1894-1904. Cerca con Google

Mostad, A., and Natarajan, S. (1990) Crystal and molecular structure of 3-nitro-4-hydroxy-phenylalanine nitrate. Z. Kristall. 193, 127-136. Cerca con Google

Musacchio, A., Saraste, M., and Wilmanns, M. (1994) High resolution crystal structure of tyrosine kinase SH3 domains complexed to proline-rich peptides. Nature Struct. Biol. 1, 546-551. Cerca con Google

Musacchio. A. (2002) How SH3 domains recognize proline. Adv. Protein Chem. 61, 211-268. Cerca con Google

Musi, V., Birdsall, B., Fernandez-Ballester, G., Guerrini, R., Salvatori, S., Serrano, L., and Pastore, A. (2006) New approaches to high-throughput structure characterization of SH3 complexes: the example of myosin-3 and myosin-5 from S. cerevisiae. Protein Sci. 15, 795-807. Cerca con Google

Myles, T., Church, F.C., Whinna, H.C., Monard, D., and Stone, S.R. (1998) Role of thrombin anion-binding exosite-I in the formation of thrombin-serpin complexes. J. Biol. Chem. 273, 31203-31208. Cerca con Google

Myles, T., Le Bonniec, B.F., and Stone, S.R. (2001) The dual role of thrombin's anion-binding exosite-I in the recognition and cleavage of the protease-activated receptor 1. Eur. J. Biochem. 268, 70-77. Cerca con Google

Naski, M.C., and Shafer, J.A. (1991) A kinetic model for the alpha-thrombin-catalyzed conversion of plasma levels of fibrinogen to fibrin in the presence of antithrombin III. J Biol Chem. 266, 13003-13010. Cerca con Google

Naski, M.C., Fenton, J.W. 2nd, Maraganore, J.M., Olson, S.T., Shafer, J.A. (1990) The COOH-terminal domain of hirudin. An exosite-directed competitive inhibitor of the action of alpha-thrombin on fibrinogen. J. Biol. Chem. 265, 13484-13489. Cerca con Google

Nicastro, G., Baumer, L., Bolis, G., and Tatò, M. (1997) NMR solution structure of a novel hirudin variant HM2, N-terminal 1-47 and N64 ? V+G mutant. Biopolymers 41, 731-749. Cerca con Google

Nilsson, B., Horne, M.K. 3rd, and Gralnick, H.R. (1983) The carbohydrate of human thrombin: structural analysis of glycoprotein oligosaccharides by mass spectrometry. Arch. Biochem. Biophys. 224, 127-1233. Cerca con Google

Nilsson, B.L., Soellner, M.B., and Raines, R.T. (2005) Chemical synthesis of proteins. Annu. Rev. Biophys. Biomol. Struct. 34, 91-118. Cerca con Google

O’Connor, S.E., and Imperiali, B. (1996) Modulation of protein structure and function by asparagine-linked glycosylation. Chem. and Biol. 3, 803-812. Cerca con Google

Pace, C.N., Vajdos, F., Fee, L. Grimsley, G., and Gray, T. (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 4, 2411-2423. Cerca con Google

Papaconstantinou, M.E., Bah, A., and Di Cera E. (2008) Role of the A chain in thrombin function. Cell Mol Life Sci. 65, 1943-1947. Cerca con Google

Pawson, T., and Nash, P. (2003) Assembly of cell regulatory systems through protein interaction domains. Science 300, 445-452. Cerca con Google

Perutz, M.F. (1970) Stereochemistry of cooperative effects in haemoglobin. Nature. 228, 726-739. Cerca con Google

Peyvandi, F., Duga, S., Akhavan, S., and Mannucci, P.M. (2002) Rare coagulation deficiencies. Haemophilia. 8, 308-321. Cerca con Google

Pineda, A.O., Carrell, C.J., Bush, L.A., Prasad, S., Caccia, S., Chen, Z.W., Mathews, F.S., and Di Cera, E. (2004) Molecular dissection of Na+ binding to thrombin. J. Biol. Chem. 279, 31842-31853. Cerca con Google

Pineda, A.O., Savvides, S.N., Waksman, G., and Di Cera, E. (2002) Crystal structure of the anticoagulant slow form. J. Biol. Chem. 277, 40177-40180 Cerca con Google

Protein Sci. 1997 3 :689-97. Biosynthetic incorporation of tryptophan analogues into staphylococcal nuclease: effect of 5-hydroxytryptophan and 7-azatryptophan on structure and stability.Wong CY, Eftink MR. Cerca con Google

Puchalski, M.M., Morra, M.J., and von Wandruszka, R. (1991) Assessment of inner filter effect corrections in fluorimetry. Fresenius J. Anal. Chem. 340, 341-344. Cerca con Google

Purcell, A.W., Aguilar, M.I., and Hearn, M.T. (1999) Probing the binding behavior and conformational states of globular proteins in reversed-phase high-performance liquid chromatography. Anal. Chem. 71, 2440-2451. Cerca con Google

Rath, A., Davidson, A.R., and Deber, C.M. (2005) The structure of "unstructured" regions in peptides and proteins: role of the polyproline II helix in protein folding and recognition. Biopolymers 80, 179-185. Cerca con Google

Richardson, J,S., Richardson, D.C. (1990). The de novo synthesis of proteins. In Proteins: Form and Function. Elsevier Trends Journal. 173-182. Cerca con Google

Richardson, J.L., Fuentes-Prior, P., Sadler, J.E., Huber, R., and Bode, W. (2002) Characterization of the residues involved in the human alpha-thrombin-haemadin complex: an exosite II-binding inhibitor. Biochemistry. 41, 2535-2542. Cerca con Google

Riordan, J.F., Sokolovsky, M., and Vallee, B.L. (1967) Environmentally sensitive tyrosyl residues. Nitration with tetranitromethane. Biochemistry 6, 358-361. Cerca con Google

Rischel, C. and Poulsen, F.M. (1995) Modification of a specific tyrosine enables tracing of the end-to-end distance during apomyoglobin folding. FEBS Lett. 374, 105-109. Cerca con Google

Rischel, C., Thyberg, P., Rigler, R., and Poulsen, F.M. (1996) Time-resolved fluorescence studies of the molten globule state of apomyoglobin. J. Mol. Biol. 257, 877-885. Cerca con Google

Rodal, A.A., Manning, A.L., Goode, B.L., and Drubin, D.G. (2003) Negative regulation of yeast WASP by two SH3 domain-containing proteins. Curr. Biol. 13, 1000-1008. Cerca con Google

Rothe, M., and Mazànek, J. (1972) Side-reactions arising on formation of cyclodipeptides in solid-phase peptide synthesis. Angew. Chem. Int. Ed. 11, 293. Cerca con Google

Russo, G., Gast, A., Schlaeger, E., Angiolillo, A., and Pietropaolo, C. (1997) Stable expression and purification of a secreted human recombinant prethrombin-2 and its activation to thrombin. Prot. Expr. Purif. 10, 214-225. Cerca con Google

Rydel, T.J., Tulinski, A., Bode, W., and Huber, R. (1991) Refined structure of the hirudin-thrombin complex. J. Mol. Biol. 221, 583-601. Cerca con Google

Rydel, T.J., Yin, M., and Padmanabhan, K.P. (1994) Crystallographic structure of human gamma-thrombin. J. Biol. Chem. 269, 22000–22006. Cerca con Google

Sadasivan, C., Yee, V.C. (2000) Interaction of the factor XIII activation peptide with alpha-thrombin. Crystal structure of its enzyme-substrate analog complex. J. Biol. Chem. 275, 36942–36948. Cerca con Google

Sawicki, E., Stanley, T.W., and Elbert, W.C. (1964) Quenchofluorometric analysis for fluoranthenic hydrocarbons in the presence of other types of aromatic hydrocarbons. Talanta 11, 1433-1441. Cerca con Google

Scacheri, E., Nitti, G., Valsasina, B., Orsini, G., Visco, C., Ferreia, M., Sawyer, R.T., and Sarmientos, P. (1993) Novel hirudin variants from the leech. Hirudinaria manillensis. Amino acid sequence, cDNA cloning and genomic organization. Eur J Biochem. 214, 295-304. Cerca con Google

Schechter, I., and Berger, A. (1967) On the size of the active site in proteases. I. Papain, Biochem. Biophys. Res. Commun. 27, 157–162. Cerca con Google

Searle, A.S., and Williams, D.H. (1992) The cost of conformational order: entropy changes in molecular association. J. Amer. Chem. Soc. 114, 10690-10697. Cerca con Google

Selvin, P.R. (1995) Fluorescence resonance energy transfer. Methods Enzymol. 246, 300-334. Cerca con Google

Sheehan, J. P., and Sadler, J. E. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5518–5522. Cerca con Google

So, I.S., Lee, S., Kim, S.W., Hahm, K., and Kim, J. (1992) Purification and activation of recombinant human prethrombin 2 produced in E. coli. Korean Biochem. J. 25, 60–65. Cerca con Google

Soejima, K., Mimura, N., Yonemura, H., Nakatake, H., Imamura, T., and Nozaki, C. (2001) An efficient refolding method for the preparation of recombinant human prethrombin-2 and characterization of the recombinant derived ?-thrombin. J.Biochem. 130, 269-277. Cerca con Google

Steen, M., and Dahlback, B. (2002) Thrombin-mediated proteolysis of factor V resulting in gradual B-domain release and exposure of the factor Xa-binding site. J. Biol. Chem. 277, 38424–38430. Cerca con Google

Steiner, R.F., Albaugh, S., and Kilhoffer, M.C. (1991) Distribution separations between groups in an engineered calmodulin. J. Fluoresc. 1, 15-22. Cerca con Google

Steiner, V., Knecht, R., Börsen, K.O., Gasssmann, E., Stone, S.R., Raschdorf, F., Schaleppi, J.-M., and Maschler, R. (1992) Primary structure and function of novel O-glycosylated hirudins from the leech hirudinaria manillensis. Biochemistry 31, 2294-2298. Cerca con Google

Stevens, W.K., and Nesheim,M.E. (1993) Structural changes in the protease domain of prothrombin upon activation as assessed by N-bromosuccinimmide modification of tryptophan residues in prothrombin-2 and thrombin. Biochemistry. 32, 2787-2794. Cerca con Google

Stoll, V.S., and Blanchard, J. (1990) Buffers: principles and practice. Methods Enzymol. 182, 24-38. Cerca con Google

Strickland, E. H. (1974) Aromatic contributions to circular dichroism spectra of proteins. CRC Crit. Rev. Biochem. 3, 113-175. Cerca con Google

Stringer, K.A., and Lindenfeld, J. (1992) Hirudins: antithrombin anticoagulants. Ann Pharmacother. 12, 1535-1540. Cerca con Google

Stubbs, M.T., Oschkinat, H., Mayr, I., Huber, R., Angliker, H., Stone, S.R., and Bode, W. (1992) The interaction of thrombin with fibrinogen: A structural basis for its specificity. Eur. J. Biochem. 206, 187-195. Cerca con Google

Szyperski, T., Güntert, P., Stone, S.R., and Wütrich, K. (1992) Nuclear magnetic resonance solution structure of hirudin (1-51) and comparison with corresponding three-dimensional structures determined using the complete 65-residue hirudin polypeptide chain. J. Mol. Biol. 228, 1193-1205. Cerca con Google

Tcherkasskaya, O., and Ptitsyn, O.B. (1999) Direct energy transfer to study the 3D structure of non-native proteins: AGH complex in the molten globule state of apomyoglobin. Protein Eng. 12, 485-490. Cerca con Google

Tong, A.H., Drees, B., Nardelli, G., Bader, G.D., Brannetti, B., Castagnoli, L., Evangelista, M., Ferracuti, S., Nelson, B., Paoluzi, S., Quondam, M., Zucconi, A., Hogue, C.W., Fields, S., Boone, C., and Cesareni, G. (2002) A combined experimental and computational strategy to define protein interaction networks for peptide recognition modules. Science 295, 321-324. Cerca con Google

Tsiang, M., Jain, A.K., and Gibbs, C.S. (1997) Functional requirements for inhibition of thrombin by Cerca con Google

Tsiang, M., Jain, A.K., Dunn, K.E., Rojas, M.E., Leung, L.L., and Gibbs, C.S. (1995) Functional mapping of the surface residues of human thrombin. J Biol Chem. 270, 16854-16863. Cerca con Google

Tsumoto, K., Ejima, D., Kumagai, I., and Arakawa, T. (2003) Practical considerations in refolding proteins from inclusion bodies. Prot. Expr. Pur. 28, 1-8. Cerca con Google

Twine, S.M., and Szabo, A.G. (2003) Fluorescent amino acid analogs. Methods Enzymol. 360, 104-127. Cerca con Google

Van Deerlin, V.M., and Tollefsen, D.M. (1991) The N-terminal acidic domain of heparin cofactor II mediates the inhibition of alpha-thrombin in the presence of glycosaminoglycans. J. Biol. Chem. 266, 20223-20231. Cerca con Google

Verhamme, I.M., Olson, S.T., Tollefsen, D.M., and Bock, P.E. (2002) Binding of exosite ligands to human thrombin. Re-evaluation of allosteric linkage between thrombin exosites I and II. J. Biol. Chem. 277, 6788-6798. Cerca con Google

Vidal, M., Gigoux, V., and Garbay, C. (2001) SH2 and SH3 domains as targets for anti-proliferative agents. Crit. Rev. Oncol. Hematol. 40, 175-186. Cerca con Google

Vidal, M., Goudreau, N., Cornille, F., Cussac, D., Gincel, E., and Garbay, C. (1999) Molecular and cellular analysis of Grb2 SH3 domain mutants: interaction with Sos and dynamin. J. Mol. Biol. 290, 717-730. Cerca con Google

Vijayalakshmi, J., Padmanabhan, K.P., Mann, K.G., and Tulinsky, A. (1994) The isomorphous structures of prethrombin-2, hirugen-, and PPACK-thrombin: Changes accompanying activation and exosite binding to thrombin. Protein Sci. 3, 2254-2271. Cerca con Google

Vindigni, A., Dang, Q.D., and Di Cera, E. (1997) Site-specific dissection of substrate recognition by thrombin. Nat. Biotechnol. 31, 11721-11730. Cerca con Google

Vindigni, A., De Filippis, V., Zanotti, G., Visco, C., Orsini, G., and Fontana, A. (1994) Probing the structure of hirudin from Hirudinaria manillensis by limited proteolysis: Isolation, characterization and thrombin-inhibitory properties of N-terminal fragments. Eur. J. Biochem. 226, 323-333. Cerca con Google

Vu, T.K., Hung, D.T., Wheaton, V.I., and Coughlin, S.R. (1991) Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell. 64, 1057-1068. Cerca con Google

Wallace, C.J. (1993). Understanding cytochrome c function: engineering protein structure by semisynthesis. FASEB J. 7, 505-515. Cerca con Google

Wang, C., Eufemi, M., Turano, C., and Giartosio, A. (1996) Influence of the carbohydrate moiety on the stability of glycoproteins. 11, 7299-7307. Cerca con Google

Wells, C.M. and Di Cera, E. (1992) Thrombin is a Na+-activated enzyme. Biochemistry 31, 11721-11730. Cerca con Google

Wells, J.A. (1990) Additivity of mutational effects in proteins. Biochemistry 29, 8509-8517. Cerca con Google

Wong, C.Y., and Eftink, M.R. (1997) Biosynthetic incorporation of tryptophan analogues into staphylococcal nuclease: effect of 5-hydroxytryptophan and 7-azatryptophan on structure and stability. Protein Sci. 3, 689-697. Cerca con Google

Workman, E.F., and Lundblad, R.L. (1978) The effect of monovalent cations on the catalytic activity of thrombin. Arch. Biochem. Biophys. 185, 544-548. Cerca con Google

Wu, P. and Brand, L. (1994) Resonance energy transfer: methods and applications. Anal. Biochem. 18. 1-13. Cerca con Google

Wu, Q.Y., Sheehan, J.P., Tsiang, M., Lentz, S.R., Birktoft, J.J., and Sadler, J.E. (1991) Single amino acid substitutions dissociate fibrinogenclotting and thrombomodulin-binding activities of human thrombin. Proc Natl Acad Sci USA 88, 6775-6779. Cerca con Google

Wu, X., Knudsen, B., Feller, S.M., Zheng, J., Sali, A., Cowburn, D., Hanafusa, H., and Kuriyan, J. (1995) Structural basis for the specific interaction of lysine-containing proline-rich peptides with the N-terminal SH3 domain of c-Crk. Structure 3, 215-226. Cerca con Google

Xenarios, I., and Eisenberg, D. (2001) Protein interaction databases. Curr Opin Biotechnol. 12, 334-339. Review. Cerca con Google

Yan, Y., and Marriott, G. (2003) Analysis of protein interactions using fluorescence technologies. Curr. Opin. Chem. Biol. 7, 635-640. Cerca con Google

Ye, J., Liu, L.W., Esmon, C.T., and Johnson, A.E. (1992) The fifth and sixth growth factor-like domains of thrombomodulin bind to the anion-binding exosite of thrombin and alter its specificity. J. Biol. Chem. 267, 11023-11028. Cerca con Google

Zarrinpar, A., and Lim, W.A. (2000) Converging on proline: the mechanism of WW domain peptide recognition. Nature Struct. Biol. 7, 611-613. Cerca con Google

Download statistics

Solo per lo Staff dell Archivio: Modifica questo record