Small protein-domain affinity reagents and D-proteins

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Antibodies are the most widely used affinity reagents, that bind with high affinity and specificity to a target protein of interest. Antibodies have been valuable tools for studying protein function and their role in cell signaling cascades, in both normal and disease states. In addition, these reagents are gaining prominence and utility in diagnosis and therapy, given that they can be used towards detection or inhibition of protein markers of disease.My research program focuses on engineering small, stable protein domains (de novo protein interfaces that can potentially recognize target proteins. However, the small size of these scaffolds presents significant technical challenges in making well-folded affinity reagents. We have previously explored several key factors in designing combinatorial libraries. Structural diversity and the amino acid composition of the mutated interface were determined to be important factors in generating affinity reagents to a wide variety of target proteins.In this proposal, we seek to study the effect of structural rigidity of the scaffold proteins on the effectiveness of generating affinity reagents. Given that molecular recognition requires a conformational fit with the target protein, we hypothesize that rigid scaffolds with minimal conformation flexibility may not be ideal. We propose a systematic study by comparing identical combinatorial libraries based on regular and stabilized versions of a given scaffold protein.We have previously reported the first use of small protein domain scaffolds to generate folded D-protein affinity reagents. D-protein affinity reagents are stable, resistant to proteolysis and non-immunogenic, making these reagents ideal for in vivo applications in molecular imaging. However, this is a nascent field, with several fundamental questions that remain unanswered. What is the fate of the cells that interact with these unnatural proteins? Do these proteins accumulate in cells following receptor-mediated endocytosis? In this proposal, we aim to address these queries by developing D-protein and L-protein affinity reagents that bind to a cell surface receptor EphA2. The proposed studies will have impact in developing effective combinatorial libraries for affinity reagent development and lay the groundwork for development of D-protein affinity reagents.