Genetically encoded non-canonical amino acids reveal asynchronous dark reversion of chromophore, backbone, and side-chains in EL222

0571540 - BTÚ 2024 RIV US eng J - Journal Article
Chaudhari, Aditya S. - Chatterjee, Aditi - Domingos, C. A. O. - Andrikopoulos, Prokopis C. - Liu, Yingliang - Andersson, Inger - Schneider, Bohdan - Lorenz-Fonfria, Victor A. - Fuertes, Gustavo
Genetically encoded non-canonical amino acids reveal asynchronous dark reversion of chromophore, backbone, and side-chains in EL222.
Protein Science. Roč. 32, č. 4 (2023), č. článku e4590. ISSN 0961-8368. E-ISSN 1469-896X
R&D Projects: GA MŠMT(CZ) EF18_046/0015974; GA MŠMT EF15_003/0000447; GA MŠMT(CZ) LM2018127
Institutional support: RVO:86652036
Keywords : flavoproteins * FTIR spectroscopy * genetic code expansion * kinetics * vis spectroscopy
OECD category: Biochemistry and molecular biology
Impact factor: 8, year: 2022
Method of publishing: Open access
https://onlinelibrary.wiley.com/doi/10.1002/pro.4590

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'alpha extension of the LOV domain in controlling EL222 photocycle length.
Permanent Link: https://hdl.handle.net/11104/0347902