Structural Studies with Neurotoxic Phospholipases A2.

Abstract

Crotoxin, the major component of venom from Crotalus snakes, is a heterodimeric complex that contains a phospholipase A2 (PLA2) and exerts a lethal action by blocking neuromuscular transmission. Crotoxin consists of a noncovalent association of an acidic, nonezymatic and nontoxic subunit, the crotapotin (or crotoxin A; CA) and a basic and toxic PLA2 subunit, the crotoxin B (CB). Crotalus neutralization factor (CNF) is an endogenous inhibitor of PLA2s isolated from Crotalus durissus terrificus plasma blood. CNF would bind only to CB and provides a protection against snake self-envenomation. This research project proposes structural and functional studies with crotoxin, its isolated subunits (CA and CB) and CNF towards the techniques of x-ray crystallography, small angle x-ray scattering (SAXS), fluorescence spectroscopy and site-directed mutagenesis. In solution (SAXS and fluorescence) and site-directed mutagenesis experiments never has been performed before with crotoxin and can provide essential information about the mechanism of action of crotoxin B and help to develop mechanisms for its inhibition. In this research project, the applicant pretends to get more understanding of in solution structural molecular biology techniques since that he obtained great experience in x-ray protein crystallography during your master and PhD. Furthermore, this research project proposes the application insertion in functional studies due to site-directed mutagenesis experiments and subsequently in vitro validation. These experiments will provide to the applicant a great autonomy to elaboration and execution of scientific research projects. Along the master and PhD, the applicant was supported by FAPESP with an excellent academic yield. The applicant has published 14 articles in addition to an article that was submitted and another two actually in preparation.