Engineering A Modular Protein Color Switch using An Entropy‐driven Beta Strand Exchange

Abstract

Listen

Translate article

Protein conformational switches are widely used in functional regulation and biosensing to visualize biological substances in vitro and in vivo. A major challenge in protein switch design strategies is coupling the target recognition domain to an output domain to produce a change in fluorescence wavelength or intensity. To overcome this challenge, we tested a rational design strategy that exploits two key protein engineering principles (1) loop entropy, by which long insertions into a loop of a host protein destabilizes the host due to an entropic cost associated with loop closure unless the inserted sequence adopts a folded structure; (2) alternate frame folding (AFF), which allows a protein (GFP variants, in this case) to switch between two mutually exclusive folds. For proof-of-concept, we chose an unfolded, circularly permuted FK506-binding protein (cpFKBP) as the input recognition domain, and inserted it in one of the two mutually exclusive folds of the GFP-AFF fusion protein. Upon addition of ligand, cpFKBP folding effects a conformational change in which the 10th beta strand of GFP exchanges, replacing Thr203 (green state) with Tyr203 (yellow state). Single point mutations and insertions were introduced to thermodynamically balance the protein switch for response within a reasonable timescale. We observe a shift in emission wavelength within an hour in response to ligand in vitro and in cellulo with a yellow:green color change of ~2.5-fold. This protein switch design has the advantages of genetic encodability, potential modularity and a ratiometric response. The design strategy further provides insight into protein engineering principles for application in molecular diagnostics, cellular biosensors and activatable proteins.