Genetically encoded non-canonical amino acids reveal asynchronous dark reversion of chromophore, backbone, and side-chains in EL222
Abstract
Photoreceptors containing the light-oxygen-voltage (LOV) domain elicit biological responses upon excitation of their flavin mononucleotide (FMN) chromophore by blue light. The mechanism and kinetics of dark-state recovery are not well understood.
Here we incorporated the non-canonical amino acid p-cyanophenylalanine (CNF) by genetic code expansion technology at 45 positions of the bacterial transcription factor EL222. Screening of light-induced changes in infrared (IR) absorption frequency, electric field and hydration of the nitrile groups identified residues CNF31 and CNF35 as reporters of monomer/oligomer and caged/decaged equilibria, respectively.
Time-resolved multi-probe UV/visible and IR spectroscopy experiments of the lit-to-dark transition revealed four dynamical events. Predominantly, rearrangements around the A'alpha helix interface (CNF31 and CNF35) precede FMN-cysteinyl adduct scission, folding of alpha-helices (amide bands), and relaxation of residue CNF151.
This study illustrates the importance of characterizing all parts of a protein and suggests a key role for the N-terminal A'α extension of the LOV domain in controlling EL222 photocycle length.