CAREER: Engineered Substrate Specificity of Serine Proteases

San Francisco State University, San Francisco CA

Investigators

Abstract

The principles that govern the mechanism(s) by which serine proteases recognize their respective substrates are only partially understood. The goal of this research project is to advance the understanding of this area using engineered derivatives of the classic serine protease, trypsin. The selected engineered derivatives are structurally identical to wild type rat trypsin except the eponymous serine, serine 195 (Ser-195) has been replaced with a threonine (Thr) residue and/or the disulfide bridge formed by cysteines (Cys) 42 and 58 has been removed by substitution with various other amino acids. The effect of the three simultaneous substitutions is to produce a functional threonine protease whose activity toward typical trypsin substrates varies as a function of (1) the size of the chemical group that occupies the position immediately C-terminal to the scissile bond in synthetic substrates and (2) the van der Waals volume of the residues at positions 42 and 58. The specific aims of this study are to (1) modify the binding site of wild type trypsin and the Ser-195 to Thr trypsin variant by substituting amino acids with various physical and chemical properties; (2) synthesize a fluorescence resonance energy transfer peptide library designed to probe amino acid preference and selectivity; and (3) characterize the variants structurally and kinetically, specifically with respect to their substrate specificities. The results from the experiments described in this project will provide insight into the molecular basis for substrate specificity in serine proteases and provide a basis for the design of new substrate specificity into trypsin-fold serine proteases. Broader Impacts Despite living in the birthplace of biotechnology and consequently an area rich in scientific resources and influence, many students in the San Francisco Bay area, particularly those who are members of underrepresented minority groups in science, have not considered a career in science or do not see a career in science as a viable possibility. One significant contributor to the lack of vision by these students could be the lack of exposure to real science and real scientists at the secondary educational level. The educational component of this Career Development plan addresses this problem by increasing the visibility of science and scientists, particularly minority scientists, in the community through the establishment of formal relationships with a local high school or schools that have significant minority student populations. During the school year, the PI will work with a high school science faculty member to design real-world molecular biology/biochemistry experiments that can be executed in the school laboratory to give students a better appreciation of what scientists actually do. Additionally, a student, or students, from the school will be selected to participate in a paid summer internship in the project. Many of the students that major in biochemistry at San Francisco State University (SFSU) have aspirations of either becoming employed in the biotech industry or pursuing advanced degrees in biotechnology related fields after graduation. Primarily undergraduate and MS level graduate students who will be trained by the principal investigator will carry out the experiments in this project. The students will gain experience in protein structure analysis, protein expression and purification, enzymatic analysis and various molecular biology techniques.