Cofactor Role in Protein Folding

Tulane University, New Orleans LA

Investigators

Abstract

Wittung-Stafshede MCB 0075902 This project aims towards probing the role of cofactors in protein-folding reactions. The targeted proteins are azurin, with a copper-ion cofactor, and flavodoxin, coordinating an organic flavin mononucleotide. Biophysical characterization by circular dichroism, fluorescence, and various biochemical methods will aid in revealing the effect of each cofactor on its corresponding protein stability and unfolded polypeptide structure. In each case, the effect on folding speed imposed by having the cofactor bound in the unfolded state will be probed by time-resolved experiments on the protein with and without the cofactor. Time-resolved experiments will be performed using both stopped-flow mixing (ms time scale) and electron-transfer triggering (ms time scale) methods, the latter technique is based on laser-triggered photochemistry. Since oxidized azurin and flavodoxin are more stable than their reduced counterparts, rapid photochemical electron extraction from the unfolded, reduced protein at suitable denaturant conditions, may trigger folding of the newly oxidized protein. Mechanisms by which proteins reach their unique three-dimensional structures are still poorly understood. To reveal pathways governing the native forms of proteins, it is important to describe conformational preferences in the unfolded proteins. Any structural preference in the unfolded state will restrict the ensemble of conformations available to the polypeptide and therefore could guide, or direct, the folding process. Residual structure in an unfolded protein may exist due to coordination of a cofactor. This project will test if cofactors, in case of two selected cofactor-binding proteins, can act as nucleation points that influence speed and/or mechanism of polypeptide-folding reactions.