Converting a Binding Protein into a Biosensing Conformational Switch Using Protein Fragment Exchange

Abstract

Biosensors can be used in applications ranging from identifying disease biomarkers to detecting spatial and temporal distributions of specific molecules in living cells. A major challenge facing biosensor development is how to functionally couple a biological recognition domain to an output module so that the binding event can be transduced to a visible and quantifiable signal [e.g., Förster resonance energy transfer (FRET)]. Most designs achieve coupling by means of a binding protein that changes conformation upon interacting with its target. This approach is limited by the fact that few proteins possess such natural allosteric mechanisms, and for those that do, the conformational change is frequently not extensive enough to produce a large change in distance between FRET donor and acceptor groups. Here, we introduce protein fragment exchange (FREX) to address both problems. FREX employs two components: a folded binding protein and a fragment duplicated from it, the latter of which can be chosen from many possible fragments. The system is rationally tuned so that addition of ligand induces a conformational change in which the fragment exchanges positions with the corresponding segment of the binding protein. Placing fluorescent donor and acceptor groups on the binding protein and fragment reduces the background level of FRET of the unbound sensor, resulting in a ratiometric FRET response that is expected to be strong and reproducible from protein to protein. FREX is demonstrated using fibronectin III, a monobody binding scaffold that has been tailored to recognize multiple targets. Sensors labeled with Alexa FRET pairs exhibit ratiometric FRET changes of up to 8.6-fold and perform equally well in buffer and serum. A genetically encoded variant of this sensor is shown to be functional in cell lysates and in mammalian cell cultures.

Synthetic protein switches: design principles and applications

“…Binding to cAbl kinase co-operatively stabilized the assembly of the FN3-based binder from two partial fragments, and was resolved by intermolecular FRET between YFP and CFP [34]. This promises to be a generic method for converting globular binding scaffolds into allosteric fluorescent sensors.…”

Construction of Allosteric Protein Switches by Alternate Frame Folding and Intermolecular Fragment Exchange

“…In the former scenario the solution is to introduce tuning mutations into the N ′-frame instead of the N- frame. The expectation is that Δ θ will initially increase and then approach a maximum value as the N ′-fold is progressively destabilized by packing mutations of increasing severity [3]. Any one of these variants can be used for sensing, although for the typical case in which a balance between high Δ θ and low K d is desired, the mutant that yields outcome (i) is selected as the final optimized product.…”

“…2. Our simulations have shown that it is possible, although highly unlikely, that a ternary complex will form in the absence of a packing mutation, as the entropic barrier for intermolecular folding is usually too large to be overcome by ligand-binding energy alone [3]. A more realistic concern is that the binary complex of full-length POI and fragment will form without ligand present.…”

“… 1 Because a portion of the binding energy is used to drive the N → N ′ conformational change, the observed K d will be greater than the intrinsic K d of the POI by a factor of (1 + K N +K N′ )/K N , where K= exp (−Δ G unfold /R.T) for the respective N- and N ′-folds [3]. …”

Influence of Secondary-Structure Folding on the Mutually Exclusive Folding Process of GL5/I27 Protein: Evidence from Molecular Dynamics Simulations

“…Such a biosensor is a protein containing duplicated segments in which only one of the two fragments at a time is incorporated in the folded protein. Stratton et al developed the alternate frame folding (AFF) strategy from a wild-type fold (N) to its circularly-permuted fold (N’) to build a switchable protein based on conformational changes [33,34,35,36,37,38]. Although MEF and AFF are incorporated into the folding-unfolding switch design, experimental studies on their switch mechanisms and the details of conformational variations at atomic level are limited so far.…”