Multiply Perturbed Response to Disclose Allosteric Control of Conformational Change: Application to Fluorescent Biosensor Design
Proteins exhibit remarkable conformational flexibility, enabling precise functional regulation through allostery. A key application of allostery is in the design of protein-based sensors, which detect environmental changes--such as ligand binding or post-translational modifications--and convert these cues into measurable signals (e.g., fluorescence). Here, we investigate a series of ligand-binding proteins that serve as sensing domains in direct-response fluorescent biosensors, wherein ligand binding enhances fluorescence output. We employ a multiple force application approach which we term Multiply Perturbed Response (MPR) to identify "hot spot" residues that drive the conformational transition from an apo (inactive/OFF) to a holo (active/ON) state. We first present two efficient computational approaches to determine residues and forces that maximize the overlap of the observed conformational change. We then determine the overlap maximizer residues for up to five force insertion locations, and we compare them with actual insertion sites used in existing biosensors. This work enhances the utility of linear response theory-based methods in uncovering multiple functionally significant regions that trigger a known conformational change. The approach might prove useful not only in the design of biosensors, or in locating allosteric sites, but may also find applications in offering physics-based collective variables in mapping conformational transition pathways of proteins.