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

Abstract

Protein conformational switches are widely used in biosensing. They are often 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 changes. 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 sixfold 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 green fluorescent protein (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 the folding of the partially disordered cpFKBP domain, to couple ligand binding to the GFP color shift. This study highlights the high-energy barriers present in GFP folding which cause β-strand exchange to be slow and are also likely responsible for the shift from the β-strand exchange mechanism in vitro to ligand-induced chromophore maturation in cells. The proof-of-concept design has the advantages of full genetic encodability and potential for modularity. The latter attribute is enabled by the natural coupling of binding and folding and circular permutation of the input domain, which theoretically allows different binding domains to be compatible for insertion into the GFP surface loop.

An adaptable, monobody-based biosensor scaffold with FRET output

“…Circular permutation was necessary to reduce the amino-to-carboxy terminal distance of YFP, so that it could be inserted into the surface loop of FN3 without unfolding FN3 and thus induce mutually exclusive folding of the YFP and FN3 domains of the fusion protein 40,41 . cpYFP was generated by permuting Clover fluorescent protein at position 195 43 and introducing F46L, F64L, V67L, and H203Y mutations 44 to change its fluorescence from green to yellow as described in our earlier work 45 . To determine whether inserting cpYFP into FN3 WDR5 affected the function of either domain, we purified the FN3 WDR5 -cpYFP fusion protein and determined it was brightly fluorescent and bound WDR5 with K d = 727 ± 53 nM (Supplemental Figure S6).…”

Engineering protein activity into off-the-shelf DNA devices

“…By contrast, AFF introduces a ligand-induced conformational change into a protein that results in one set of residues being replaced by another. This fold shift has been used to switch on and off nuclease activity ( Karchin et al., 2017 , Mitrea et al., 2010 ), calcium and ribose binding ( Karchin et al., 2017 ; Stratton et al., 2008 ), and fluorescence ( Do and Boxer, 2013 ; John et al., 2022 ).…”

“…The foremost limitation of our current design is its slow turn-on half-time (8.25 h), which can be several orders of magnitude longer than that of split nLuc sensors ( Ni et al., 2019 ). Experimental ( John et al., 2022 ; Stratton and Loh, 2010 ) and computational ( DeGrave et al., 2018 ) studies of other AFF-modified proteins suggest that slow switching is due to a large kinetic barrier to the unfolding of the N frame. Introducing mutations to destabilize the N fold can accelerate the overall switching rate.…”

Enhancing response of a protein conformational switch by using two disordered ligand binding domains

“…The phenomenon of binding-induced folding is widespread in natural and engineered proteins alike, suggesting our design may be generalizable. Indeed, previous examples of related AFF switches have used proteins that naturally bind ligands (Karchin et al, 2017;Gräwe and Merkx, 2022;John et al, 2022), proteins engineered to bind a specific ligand (Cutler et al, 2009), and even transcription factors to drive a fold shift (Ha et al, 2006;. Moreover, although both binding domains recognized rapamycin in the present study, it is possible to use two proteins that target different ligands, allowing synergy to be programmed into the AFF design.…”

“…The second regulatory mechanism, named entropy switching (John et al, 2022), is based on the principle of loop-closure entropy (Minard et al, 2001;Scalley-Kim et al, 2003) (LCE). A circular permutant of the same unstable FKBP domain as above (cpFKBP) is inserted in between the shared and duplicated segments of the CP-fold of Bn-AFF (Figure 1C), where it acts as a surface loop in the CP-fold.…”