Improving and Understanding the Folding of Disulfide Containing Proteins

Florida International University, Miami FL

Investigators

Abstract

With the support of the Organic Dynamics Program in the Chemistry Division, Professors Watson J. Lees at Florida International University will examine the rational design and application of non-traditional thiols that will significantly increase the rate of in vitro protein folding. These non-traditional thiols will be designed using the enzyme protein disulfide isomerase (PDI) as a guide. PDI is the enzyme in humans that is reported to catalyze the folding of disulfide containing proteins. PDI itself is rarely, if ever, used for folding due to cost and low specific activity. Because PDI has a low pKa thiol in its active site and displays enhanced reactivity towards protein disulfides, aromatic thiols were selected. These also possess low thiol pKa values and enhanced reactivity towards protein disulfides. Preliminary results have demonstrated that the addition of aromatic thiols significantly increases the folding rate of disulfide containing proteins, such as ribonuclease A (RNase A), in vitro. However, before designing more complex compounds several strategies that have the potential to further improve protein-folding rates will be investigated. The first strategy involves determining an equation to predict folding rates for a particular protein under a variety of conditions so that the optimal conditions can be selected. Dramatic increases in protein folding over traditional methods are expected based on preliminary results. The Organic and Macromolecular Chemistry Program in the Chemistry Division supports Professor Watson J. Lees at Florida International University who proposes to facilitate the production of disulfide containing proteins under a variety of conditions and to understand the protein folding process. Many pharmaceutically relevant proteins and numerous extracellular proteins contain essential disulfide bonds. Common examples include insulin, human growth hormone, and erythropoietin (EPO), a glycoprotein hormone produced primarily by cells of the peritubular capillary endothelium of the kidney that is responsible for the regulation of red blood cell production. In addition to providing a greater understanding of the folding of disulfide containing proteins at a fundamental level, Professor Lees' research will also provide a valuable mechanism for training graduate students, postdoctoral fellows and undergraduate students in biochemistry and organic chemistry.