Large enhancement of response times of a protein conformational switch by computational design

The design of protein conformational switches—or proteins that change conformations in response to a signal such as a ligand binding—has great potential for developing novel biosensors, diagnostic tools, and therapeutic agents. Among the defining properties of such switches, the response time has been the most challenging to optimize. Lillian Chong and colleagues have applied a computational design strategy in synergistic combination with biophysical experiments to rationally enhance the response time of an engineered protein-based Ca2+ sensor, the calbindin-AFF switch.

Lillian Chong and her colleagues have used WE (weighted ensemble) strategy in conjunction with residue-level models to greatly extend the computational reach of molecular simulations. This approach makes it feasible to generate, within days to a few weeks using a typical computer cluster, protein conformational switching pathways as slow as tens of seconds, thereby facilitating a close interplay of simulation and experiment. Based on the simulated pathways, they have identified previously untested mutations that are promising for enhancing the response time of the switch via preferential destabilization of the ground states relative to the transient states. In addition, they have identified a negative control mutation that was predicted to have no effect on the response time via similar destabilization of the ground states relative to the transient states. As predicted, the negative control had little effect on the response times of the switch in both the forward (N → N′) and reverse (N′ → N) directions while all of the promising mutations substantially improved the response times of the switch in both directions. The largest improvement amounted to 32-fold, reducing the response time of the switch in the reverse direction from a mean first passage time of 590 to 19 ms, which is in range of the most rapid physiological Ca2+ fluctuations. The most effective pair of mutations, F50′A/ F66A, simultaneously increased the rate constants for both the N → N′ and N′ → N switching processes by more than an order of magnitude.

This computational design strategy is a general one that can be applied to any protein conformational switch of a similar size (e.g., less than a few hundred amino acids) provided that the switching process occurs on the timescale of <100 s and structures of the switch components are available from either experiment or homology modelling. Furthermore, since all protein conformational switches function based on the relative stabilities of alternate conformations, our strategy is applicable to all such switches, including ones that function by other mechanisms that do not involve as large conformational transitions as the mutually exclusive folding of protein domains, provided that the expected relative stabilities are reproduced. The strategy could, therefore, be valuable for a variety of applications, including the design of more rapid biosensors and temporally accurate control mechanisms in synthetic biology.

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