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'α helix interface (CNF31 and CNF35) precede FMN‐cysteinyl adduct scission, folding of α‐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.

Machine Learning-Assisted Engineering of Light, Oxygen, Voltage Photoreceptor Adduct Lifetime

“…As outlined in the introduction, dark recovery, as a whole, is a complex, multistep process that cannot be captured by a single technique. − As is the case for many protein engineering campaigns that rely on screening a larger number of variants, our study shows important limitations that have implications for the design of optogenetic tools based on the identified mutants. First, in this work, we only monitored the recovery of the dark-state flavin absorption by UV/Vis spectrophotometry as a proxy for the recovery of the photoreceptor, being well aware that photoreceptor dark recovery also involves recovery of the dark-state structure of the protein, which cannot be monitored by simple UV/Vis spectrophotometry.…”

“…This process, depending on the photoreceptor, results in an unfolding/unwinding of N- and C-terminal helices outside the sensory LOV domain, the dissociation of adjacent domains, or changes in the oligomerization state, ,,, which in turn mediates various physiological responses. ,− The photocycle is thermally reversible in the dark, with the recovery process involving the breaking of a covalent FMN-cysteinyl thiol bond, the deprotonation of the flavin N5 atom, as well as the structural reversal of the above-described light-induced structural changes of the photoreceptor. While FMN-Cys adduct formation is completed within microseconds (see e.g., − ), the dark recovery process can last from seconds to days, depending on the LOV protein. ,− Recent studies, e.g., using time-resolved small-angle X-ray scattering techniques, have shown that the dark recovery (described by the signaling-state lifetime τ rec ), as a whole, is a complex, multistep process that cannot be simply captured by a single technique, − and a differentiation between the dark recovery of the FMN absorbance (τ FMN ) as a proxy of FMN-Cys adduct rupture and the reversal of structural changes is necessary. Due to the huge variability in kinetics found in natural LOV photoreceptor systems, the dark-recovery process is of fundamental interest from a biophysical and biochemical perspective.…”

Regulation of IL-24/IL-20R2 complex formation using photocaged tyrosines and UV light

“…First, the suppression efficiencies are variable, context-dependent and may result in severe drops of protein yields, particularly in the case of multi-site incorporation (Amiram et al, 2015). Second, the introduction of non-native sidechains may affect protein folding, stability, binding and dynamics (Rogers et al, 2018;Kesgin-Schaefer et al, 2019;Chaudhari et al, 2023). Third, due to the usually large interfacial areas that are stabilized by multiple non-covalent interactions (hydrogen bonds, hydrophobic interactions, etc.…”

“…Obtaining "difficult" proteins requires multiple expression attempts (Peleg and Unger, 2012) or cumbersome and cost-ineffective expression systems (Hopkins et al, 2010). On top of these effects, the incorporation of ncAA may cause further decreases in protein yields and alter protein stability or function in ways that are difficult to predict (Chaudhari et al, 2023). Indeed, all our proteins of interest are naturally N-glycosylated and bear disulfide bonds, which make them hard to produce in traditional E. coli expression systems.…”