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Determination of Maromolecular Structures Using Synchrotron Radiation for the Elucidation of Enzymatic Activities

Abstract

The focus of this project is the design, construction and operation of a dedicated experimental station for protein crystallography at the The Brazilian Synchrotron Light Laboratory - LNLS, and its application in the structural studies of several proteins. In the original proposition, the objective was the determination of the structure of the enzyme Glucosamine-6-phosphate Deaminase from E. coli, which, having been already solved at IFSC/USP, opened the path for the broadening of applications of the bemaline PCr to several other projects, from the Laboratory of Protein Crystallography and Structural Biology of IFSC/USP and other research groups in Protein Crystallography in the country. The beamline is its final phase of construction and commissioning and will include cylindrical mirror and bent crystal monochromator focusing the high flux of synchrotron radiation in the horizontal plane at the position of the sample. The monochromatic radiation will be tuneable between 2.0 A to 1.0 A with the optimum wavelength at 1.6 A, chosen with the aim of maximising the photon flux from the bending magnets of the storage ring (1.37GeV). Diffraction images will be recorded on a commercial image plate detector system with on-line readout. The beamline set-up will include cooler/chillier of the samples and biochemical lab for crystallisation, heavy metal soaks, crystal storage and mounting, with thermal stable rooms at 22C and 4C. The facility, intended to serve the national and international community, is planned to be opened for external users in September/97. More than 30 structural projects, from proteins that have already been crystallized or are in the phase of crystallization experiments, have been proposed for the use of the beamline, originating from several labs, and covering studies of enzymes, plant proteins, toxins, structure based drug and vaccine design, therefore focusing in applications that are of high social and national interests. It is foreseen that the commissioning of the first protein crystallography beamline in Latin America will boost the number of protein structures determined locally and will increase the general interest from the molecular biology and biochemical research community of Brazil in this area. (AU)