Multi-State Computational Design and Experimental Characterization of Triosephosphate Isomerase Flexible Hinges

Traditional computational protein design calculations enable the evaluation and selection of protein sequences in the context of a single native structure. However many proteins natively require multiple distinct conformations for functionality. Recently, a novel algorithm has been developed which considers several structures in a single optimization calculation. This new method could be useful for designing proteins that need to accommodate several conformations. We sought to test this algorithm on triosephosphate isomerase (TIM). Loop 6 in TIM is a lid with open and closed conformations that facilitate substrate binding, product diffusion, and catalysis. It uses two 3-amino acid hinges that provide the flexibility for the open-closed transition. In the present work we use multi-state design (MSD) methods to design the flexible hinges in TIM. We are in the process of assessing the applicability of MSD for the design of flexible protein hinges by exhaustive screening of computationally designed libraries. The results will suggest whether, by considering both conformations of the enzyme instead of purely the open or closed conformations, our methods provide libraries enriched in catalytically active TIMs.