Why a Single Mutation Dims the Light: Local Chromophore Organization Modulates Fluorescence in smURFP.

Predicting mutation-induced photophysical changes in bilin-binding fluorescent proteins remains challenging. Here, we investigate the origin of fluorescence modulation in smURFP and its low-fluorescent-quantum-yield Y56R variant using classical MD simulations and QM/MM calculations. Comparison of multiple chromophore binding states supports a wild-type configuration involving A-ring attachment and chromophore reduction, whereas Y56R adopts the opposite orientation, revealing substantial plasticity of the chromophore pocket in response to a single mutation. The experimentally observed differences in fluorescence efficiency and absorption energy arise not from simple global rigidification, but from redistribution of local steric confinement and electrostatic polarization across the biliverdin scaffold. Overall, our results highlight how spatially resolved steric and electrostatic descriptors extracted from classical MD simulations can provide mechanistic insight into biliprotein photophysics.