Designer Proteases for Complete Proteomics

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Intellectual Merit: The goal of the project is to design proteases from alpha-lytic protease (aLP) that have new and different substrate specificities. The project will develop new methods and tools to determine the primary structure of proteins, in particular to determine which amino acids are post-translationally modified. Nearly all large-scale protein sequencing experiments are currently performed using mass spectrometry to analyze pieces of proteins that were digested with the protease, trypsin, which cleaves after lysine (K) and arginine (R) residues. Trypsin cleavage produces enough peptides to provide sufficient unique protein sequence for protein identification, but more complete coverage of protein sequences is required for post-translational modification (PTM) localization. Preliminary results show that aLP is ideal for proteomics because a few active site mutations alter its substrate specificity towards either small aliphatic residues or large hydrophobic residues. These "orthogonal" types of amino acids (compared to K and R, which are charged) allow access to more and different parts of the protein sequence. Building on the aLP platform, a family of engineered proteases will be designed with varying substrate cleavage specificities to enable coverage of entire protein sequences. An application that will be investigated is the determination of which sites in proteins are modified with the small ubiquitin-like modification (SUMO). The generated mutants provide ideal substrate specificity for detection of SUMOylation in the S. pombe yeast proteome. Cleavage of SUMOylated proteins using a mutant aLP generates GG-tagged lysines that can be detected with existing approaches. Thus aLP will provide the first comprehensive survey of which proteins are SUMOylated in yeast.Broader Impacts: The broader impacts of the project include development of novel proteases that will be widely useful to the proteomics community. A survey of researchers engaged in proteomics studies reveals the dire need for such robustly active "orthogonal" proteases. The project will result in large proteomic data sets that will aid in the prediction of modification sites and provide information for basic research into cell biology and protein structure/function. Data analysis programs will also be developed that will be widely disseminated via the web. Two undergraduate researchers will be involved in the project throughout the project period. The PI has a long history of mentoring undergraduates, who have entered careers in science, with a particular focus on encouraging diversity. The PI founded at UCSD the Research Scholars program that provides research training for high school students from diverse backgrounds. The participants are mentored by assigned pairs of faculty and graduate students. Mentoring is continued up to the point where participants choose colleges and majors. In the first two years, more than 90% of the students completed the program having decided on a career in scientific research. One and possibly 2 students from the Research Scholars Program will work on aspects of this NSF project.