Rational design framework for fluorescent biosensors from periplasmic binding proteins.

Periplasmic binding proteins (PBPs) are attractive scaffolds for fluorescent biosensors because they undergo large ligand-induced conformational changes and exhibit high specificity. Despite their potential, generalizable design strategies have been hindered by an incomplete understanding of how labeling positions influence protein dynamics and signal transduction. Here, we established a systematic experimental framework using the lysine-arginine-ornithine binding protein (LAO) as a model to characterize PBP-based fluorescent biosensors. Seven positions classified as endosteric, peristeric, or allosteric relative to the binding site were labeled with monobromobimane (mBBr). Our characterization revealed position-dependent effects on quantum yields, fluorescence intensity changes, ligand-binding affinities, and thermal stability. Molecular dynamics simulations (MD) of wild-type and mBBr-labeled variants provided a mechanistic context for these experimental observations. Functional biosensor positions (D51C, D53C, K228C, Y230C, and E167C) maintained open-like conformations necessary for signal generation upon ligand encounter. In contrast, peristeric position A89C, located at the hinge region, spontaneously adopted a closed-like conformation even in the absence of ligand, resulting in an inverse fluorescence response and reduced affinity. These results demonstrate that while structural criteria can guide initial site selection, experimental validation remains essential, as static structures alone cannot predict the complex interactions among labeling position, dynamics, and biosensor function. The experimental and computational framework established here with LAO as a model system provides methodological principles that may inform biosensor engineering from other PBP scaffolds, while emphasizing that conformational dynamics must be considered alongside static structural criteria.