Engineering A Fluorescent Protein Color Switch Using Entropy-driven Beta Strand Exchange

Abstract

Protein conformational switches are widely used in biosensing. They are typically composed of an input domain (which binds a target ligand) fused to an output domain (which generates an optical readout). A central challenge in designing such switches is to develop mechanisms for coupling the input and output signals via conformational change. Here, we create a biosensor in which binding-induced folding of the input domain drives a conformational shift in the output domain that results in a 6-fold green-to-yellow ratiometric fluorescence change in vitro, and a 35-fold intensiometric fluorescence increase in cultured cells. The input domain consists of circularly permuted FK506 binding protein (cpFKBP) that folds upon binding its target ligand (FK506 or rapamycin). cpFKBP folding induces the output domain, an engineered GFP variant, to replace one of its β-strands (containing T203 and specifying green fluorescence) with a duplicate β-strand (containing Y203 and specifying yellow fluorescence) in an intramolecular exchange reaction. This mechanism employs the loop-closure entropy principle, embodied by folding of the partially disordered cpFKBP domain, to couple ligand binding to the GFP color shift. This proof-of-concept design has the advantages of full genetic encodability, ratiometric or intensiometric response, and potential for modularity. The latter attribute is enabled by circular permutation of the input domain.

Engineering a Fluorescent Protein Color Switch Using Entropy-Driven β-Strand Exchange

“…These characteristics confer modularity and may aid in the development of broadly applicable engineered protein switches. 40 ■ ASSOCIATED CONTENT * sı Supporting Information…”

“…In addition to its application in cellular sensing, the findings from this study can expand the protein engineers’ toolbox as it provides insights into how to create custom input and output functions by using a combination of protein engineering techniques: AFF of the output domain and circular permutation of the input domain. These characteristics confer modularity and may aid in the development of broadly applicable engineered protein switches …”