Research Starter Grant: Engineered Substrate Specificity of Serine Proteases

San Francisco State University, San Francisco CA

Investigators

Abstract

Project Summary The principles that govern the mechanism(s) by which serine proteases recognize their respective ubstrates are only partially understood. The goal of this research project is to advance the understanding of this area using a novel engineered derivative of the classic serine protease, trypsin. The engineered derivative is identical to wild type rat trypsin except that the eponymous serine, serine 195 (Ser-195) has been replaced with a threonine (Thr) residue and the disulfide bridge formed by cysteines (Cys) 42 and 58 has been removed by substitution of those amino acids with either alanine or valine residues. 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 (the P1. position) and (2) the van der Waals volume of the residues at positions 42 and 58 (the S1. binding site). This observation suggests that, in the presence of Thr-195, the chemical and physical properties of the amino acid residues in the S1. binding site may influence the selection and subsequent hydrolysis of a given substrate with respect to its P1. residue. Additionally, the S1. subsite may be engineered such that the selection of a substrate with a given P1. residue may be selectively hydrolyzed. Therefore, the specific aims of this study are to (1) modify the S. (S-prime) binding site of wild type trypsin and the Ser-195 to Thr trypsin variant by substituting amino acids with various physical and chemical properties at positions 42 and 58 and (2) characterize the variants kinetically and with regards to their substrate specificities. Specifically, peptide substrates will be designed and synthesized to evaluate the effect of the substitutions made on P1. selectivity. By introducing such changes into the S1. binding site of the engineered threonine protease, it is expected that the variants will display preferential hydrolytic activity toward substrates whose P1. amino acid is complementary in chemical and physical nature to the amino acids used to modify the S1. binding site. The results from the experiments described in this project will provide insight into the molecular basis for extended substrate specificity in serine proteases and provide a basis for the design of substrate specificity into trypsin-fold serine proteases. Broader Impact The experiments outlined in the project will be carried out primarily by undergraduate and Master.s level graduate students that will be trained by the principal investigator. The students will gain experience in protein structure analysis, protein expression and purification, enzymatic analysis and various molecular biology techniques including PCR, mutagenesis and gene subcloning. The work will be carried out at San Francisco State University, an ethnically diverse University, providing a means for the P.I. to recruit and train students from ethnic minorities that are underrepresented in the sciences with the intention of exposing them to opportunities to pursue careers in science and preparing them to succeed in such pursuits.