Applied Crystallography

Biography

Biography: Claudia Bravo-Chaucanés

Abstract

Although fungal infections contribute substantially to human morbidity and mortality, the impact of these diseases on human health is not widely appreciated. Cryptococcosis is caused by Cryptococcus neoformans, an encapsulated fungus that causes lung disease and can spread widely in the brain and skin, affects about one million people each year and kills about 650,000. The virulence of this fungus is mediated by several factors, the antioxidant defense mechanisms have been shown to be important not only for resistance to reactive species but also for survival in the mammalian host. These antioxidant enzymes important for virulence may serve as excellent targets for antifungal therapy. The classical thioredoxin system is formed by thioredoxin reductase and its characteristic substrate the redox active protein thioredoxin, whose reduction is supported by NADPH. Thioredoxins are low molecular weight proteins that can be localized in the cytoplasm, in the mitochondria as well as in the extracellular space. Trx possess an active site made up of two cysteines in a conserved motif, CXXC, which is highly conserved. In this study, heterologous expression, purification, enzymatic characterization and crystallographic structure of oxidized Trx1 has been solved at 1.8Ã… resolution in the fungal pathogen C. neoformans. For crystallization assays, Trx1 has been concentrated to 9 mg mL-1 in 1,7 M Ammonium sulfate and 0,02 M Tris hydrochloride pH 8,5, the crystallization trials have been performed using the hanging-drop vapor diffusion method. X-ray diffraction data were collected using a synchrotron radiation source and the structure was solved by molecular replacement and refined to a crystallographic R factor of 15.0% and a free R factor of 19%.