PROJECT SUMMARY/ABSTRACT We propose to explore the use of proline analogs to engineer and probe the biophysical behavior of insulin. Insulin contains a single proline residue – at position 28 of the B-chain – which is known to play critical roles in controlling the rates of onset of action and fibrillation of pharmaceutical preparations of the protein. Replacement of proline through conventional mutagenesis has led to FDA-approved rapid- acting insulins, but destabilizes the protein with respect to fibrillation. Conventional mutagenesis suffers from a fundamental limitation when applied to proline; any amino acid change converts the conformationally restricted cyclic proline residue to a more flexible acyclic one. We have recently found that replacement of the proline residue at position 28 of the insulin B-chain by (4S)-hydroxyproline – through non-canonical amino acid mutagenesis – yields an active form of insulin that dissociates more rapidly, and fibrillates more slowly, than the wild-type protein. This approach allows one to alter critical molecular interactions around position B28 without sacrificing the unique conformational properties of proline. This proposal seeks to expand the known ‘proline chemical space’ that can be accessed in the bacterial expression of recombinant insulin. We will accomplish this objective by assessing the translational activity of a carefully chosen set of proline analogs in E. coli, by creating new prolyl-tRNA synthetases to activate the analogs of interest, and by analyzing the biophysical behavior of the resulting insulin variants by experimental and computational means. The proposed work will provide new forms of insulin with altered biophysical properties, expand the toolkit for engineering protein structure and function, and enhance our understanding of protein association and dynamics.