Structural studies of the proteins using synchrotron light

• Grant number: 99/03387-4
• Support Opportunities: Research Projects - Thematic Grants
• Start date: October 01, 1999
• End date: September 30, 2005
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Igor Polikarpov
• Grantee: Igor Polikarpov
• Host Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil
• Pesquisadores principais: Jörg Kobarg

Abstract

At present protein crystallography is the most powerful method of protein structure solution at atomic or almost atomic resolution. The three-dimensional structures of macromolecules contribute to understanding of various cell processes, such as enzymatic catalysis, immunologic response, viral infection, cellular division, transmission of genetic information, etc. In addition to that, the knowledge of protein structures is essential for the rational drug design and genetic modification of proteins. Brazil is the only country in Southern Hemisphere that bas a synchrotron storage ring and a dedicated synchrotron radiation beamline. The beamline, that began to function little over a year-and-a-half ago, has a very high X-ray flux and already was used to tackle over 60 different crystallographic projects from Brazil and other countries (Argentina, Mexico, Russia, Germany, Australia, USA). The main interest of the current project is to develop methods and equipment for protein structure determination with use of synchrotron light, as well as to optimize these methods for particular applications at the Protein Crystallography Beamline of LNLS. This would serve to turn macromolecular structure solution easier and faster. Optimization of the native and derivative data collection that includes multiple/single isomorphous replacement with anomalous scattering (MIRAS/SIRAS)and multiple/single wavelength anomalous diffraction (MADISAD) data collection will be applied in practical cases of 15 different crystallographic sub-projects. These sub-projects will be persuaded using molecular replacement (II.1, II.2, II.3, II.5-8, II.12- II.15, and, possibly, II.4), isomorphous replacement and anomalous diffraction techniques (II.3, II.4, II.9- II.11). Atomic resolution diffraction studies will be conducted within subproject II.14 and, probably other sub-projects. Cryo-crystallographic techniques are being optimized for use at protein crystallographic beamline at LNLS and will be used throughout the project implementation. Structural sub-projects are focussed on the proteins related to human health (II.1, II.3, II.4, II.6), virus infection (II.5), cancer treatment (II.2), inflammation processes (II.7), venom poisoning (II.14, II.15); industrial applications (II.9- II.11); basic biological studies of glycosilated proteins (II. 1, II.8-II. 11) and glycolitic pathway enzymes (II.12, II. 13). Proteins in study ranges from small non-glycosilated polipeptides (7.5 kDa) to large heavily glycosilated molecules (~100 kDa, glycosilation of about 15%).Synchrotron radiation techniques are a pivot axis and joining force of the projects. Many of them would be difficult, if not impossible, to realize without use of synchrotron light. Development and adaptation of modern synchrotron radiation techniques as applied to structural molecular biology and their thorough testing on a large number of crystallographic projects will significantly increase the speed and quality of data collection and structure solution. This will lay a basis for future structural genomics initiatives and rational drug design studies. Technical advances achieved within the course of the project will be beneficial for all macromolecular crystallographic community of Brazil and Mercosul. The wealth of structural information gathered during the project execution might provide new insights into important health, medical and industrial problems.The project is divided into two parts: Methodological Studies and Structural Studies. In Methodological Studies part we plan to optimize native and derivative data collection for synchrotron radiation macromolecular structure solutions.In Structural Studies Part we present 15 projects directed toward crystal structure solution and comparative analysis of a number of biologically important proteins. (AU)

Scientific publications

(10)

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

SANTOS, MARIA A. M.; SANTOS, SILVIA M.; MATOZO, HUITA C.; PORTUGAL, RODRIGO V.; IULIANO, RODOLFO; FUSCO, ALFREDO; POLIKARPOV, IGOR. Expression, purification, and characterization of rat protein tyrosine phosphatase eta catalytic domain. Protein Expression and Purification, v. 41, n. 1, p. 113-120, May 2005. (99/03387-4)

RAZZERA, GUILHERME; VERNAL, JAVIER; PORTUGAL, RODRIGO V.; CALGARO, MARCOS R.; FERNANDEZ, PABLO; ZAKIN, MARIO M.; POLIKARPOV, IGOR; TERENZI, HERNÁN. Expression, purification, and initial structural characterization of rat orphan nuclear receptor NOR-1 LBD domain. Protein Expression and Purification, v. 37, n. 2, p. 443-449, Oct. 2004. (99/03387-4)

AMBROSIO, ANDRE L. B.; DIAS, SANDRA M. G.; POLIKARPOV, IGOR; ZURIER, ROBERT B.; BURSTEIN, SUMNER H.; GARRATT, RICHARD C.. Ajulemic acid, a synthetic nonpsychoactive cannabinoid acid, bound to the ligand binding domain of the human peroxisome proliferator-activated receptor gamma. Journal of Biological Chemistry, v. 282, n. 25, p. 18625-18633, June 2007. (06/00182-8, 98/14138-2, 99/03387-4)

ROJAS, A. L.; NAGEM, R. A. P.; NEUSTROEV, K. N.; ARAND, M.; ADAMSKA, M.; ENEYSKAYA, E. V.; KULMINSKAYA, A. A.; GARRATT, R. C.; GOLUBEV, A. M.; POLIKARPOV, I.. Crystal structures of beta-galactosidase from Penicillium sp. and its complex with galactose. Journal of Molecular Biology, v. 343, n. 5, p. 1281-1292, Nov. 2004. (99/03387-4)

KRAUCHENCO‚ S.; NAGEM‚ R.A.P.; DA SILVA‚ J.A.; MARANGONI‚ S.; POLIKARPOV‚ I.. Three-dimensional structure of an unusual Kunitz (STI) type trypsin inhibitor from Copaifera langsdorffii. Biochimie, v. 86, n. 3, p. 167-172, 2004. (99/03387-4)

MATOZO‚ H.C.; SANTOS‚ M.A.M.; DE OLIVEIRA NETO‚ M.; BLEICHER‚ L.; LIMA‚ L.M.T.R.; IULIANO‚ R.; FUSCO‚ A.; POLIKARPOV‚ I.. Low-Resolution Structure and Fluorescence Anisotropy Analysis of Protein Tyrosine Phosphatase [eta] Catalytic Domain. BIOPHYSICAL JOURNAL, v. 92, n. 12, p. 4424-4432, 2007. (99/03387-4, 06/00182-8)

NASCIMENTO, ALESSANDRO S.; KRAUCHENCO, SANDRA; GOLUBEV, ALEXANDER M.; GUSTCHINA, ALLA; WLODAWER, ALEXANDER; POLIKARPOV, IGOR. Statistical coupling analysis of aspartic proteinases based on crystal structures of the Trichoderma reesei enzyme and its complex with pepstatin A. Journal of Molecular Biology, v. 382, n. 3, p. 763-778, Oct. 2008. (99/03387-4)

MARTINS, NÁDIA HELENA; CALAZANS, ALEXANDRE; BRINDEIRO, RODRIGO DE MORAES; TANURI, AMILCAR; ANTUNES, OCTAVIO AUGUSTO CEVA; POLIKARPOV, IGOR; SANCHES, MARIO. Crystallization of a non-B and a B mutant HIV protease. ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, v. 60, n. 9, p. 1625-1627, Sept. 2004. (99/03387-4)

KRAUCHENCO, SANDRA; MARTINS, NADIA H.; SANCHES, MARIO; POLIKARPOV, IGOR. Effectiveness of commercial inhibitors against subtype F HIV-1 protease. Journal of Enzyme Inhibition and Medicinal Chemistry, v. 24, n. 3, p. 638-645, 2009. (99/03387-4, 06/00182-8)

NAGEM, RONALDO ALVES PINTO; COLAU, DIDIER; DUMOUTIER, LAURE; RENAULD, JEAN-CHRISTOPHE; OGATA, CRAIG; POLIKARPOV, IGOR. Crystal structure of recombinant human interleukin-22. Structure, v. 10, n. 8, p. 1051-1062, Aug. 2002. (99/03387-4)