Collaborative Research: Towards a General Design Approach to Arrest Non-Native Aggregation of Multi-Domain Proteins

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0853543FernandezIntellectual Merit - Non-native aggregation is a ubiquitous hurdle to successful over-expression and purification of recombinant proteins throughout the biotechnology and biopharmaceutical industries. This is particularly the case for multi-domain proteins, such as natural and designed antibodies, as well as for other predominantly-beta proteins. Fundamentally, rational design of these more complex proteins is handicapped by limitations in the mechanistic understanding of key features of sequence and structure that control aggregation of multi-domain proteins. This project seeks to lay a foundation both to fill the gaps in fundamental understanding of the mechanism(s) of multi-domain protein aggregation, and to provide first-generation design rules to imbue aggregation resistance. Human gamma-D Crystallin (gamma D-Crys) will be the model multidomain protein, chosen because it possesses a number of desirable features: (1) it is a single chain, two domain protein; (2) it represents an important set of all-beta proteins; (3) it has established aggregation and folding behavior; (4) it has available, aggregation-prone mutants. Broader Impact - The proposed research will result in an improved mechanistic understanding of aggregation and rational design of aggregation resistance for multi-domain, all-beta Greek-key proteins, of which a number of antibody constructs are a subclass of widespread biological and biotechnological interest. The research will also provide a framework for the education and training of graduate and undergraduate students in the PIs' laboratories, with a specific focus on cutting-edge experimental and modeling tools. As in the past, the PIs will involve students drawn from underrepresented groups in science and engineering. Finally, a set of examples and problems will be developed to incorporate aspects of this research into the undergraduate curricula, including computational and modeling activities. This module will be disseminated to other faculty via a web-based repository of educational materials developed at San Jose State University.