Chromophore carbonyl twisting in fluorescent biosensors encodes direct readout of protein conformations with multicolor switching

Fluorescent labeling of proteins is a powerful tool for probing
structure-function relationships with many biosensing applications.
Structure-based rules for systematically designing fluorescent biosensors
require understanding ligand-mediated fluorescent response mechanisms
which can be challenging to establish. We installed thiol-reactive
derivatives of the naphthalene-based fluorophore Prodan into bacterial
periplasmic glucose-binding proteins. Glucose binding elicited paired
color exchanges in the excited and ground states of these conjugates.
X-ray structures and mutagenesis studies established that glucose-mediated
color switching arises from steric interactions that couple protein
conformational changes to twisting of the Prodan carbonyl relative to its
naphthalene plane. Mutations of residues contacting the carbonyl can
optimize color switching by altering fluorophore conformational equilibria
in the apo and glucose-bound proteins. A commonly accepted view is that
Prodan derivatives report on protein conformations via solvatochromic
effects due to changes in the dielectric of their local environment. Here
we show that instead Prodan carbonyl twisting controls color switching.
These insights enable structure-based biosensor design by coupling
ligand-mediated protein conformational changes to internal chromophore
twists through specific steric interactions between fluorophore and
protein.