The amino acids surrounding the flavin 7a-methyl group determine the UVA spectral features of a LOV protein

Abstract

Flavin-binding light, oxygen, and voltage (LOV) domains are UVA/blue-light-sensing protein units that form a reversible flavin mononucleotide-cysteine adduct upon light induction. In their dark-adapted state, LOV domains exhibit the typical spectral features of fully oxidized riboflavin derivatives. A survey on the absorption spectra of various LOV domains revealed that the UVA spectral range is the most variable region (whereas the absorption band at 450 nm is virtually unchanged), showing essentially two distinct patterns found in plant phototropin LOV1 and LOV2 domains, respectively. In this work, we have identified a residue directly interacting with the isoalloxazine methyl group at C(7a) as the major UVA spectral tuner. In YtvA from Bacillus subtilis, this amino acid is threonine 30, and its mutation into apolar residues converts the LOV2-like spectrum of native YtvA into a LOV1-like pattern. Mutation T30A also accelerates the photocycle ca. 4-fold. Together with control mutations at different positions, our results experimentally confirm the previously calculated direction of the transition dipole moment for the UVA ππ* state and identify the mechanisms underlying spectral tuning in the LOV domains.

Proton Transfer to Flavin Stabilizes the Signaling State of the Blue Light Receptor Plant Cryptochrome

“…2). The position of the absorption band in the UVA range is a sensitive indicator for the hydrogen bonding interactions in the flavin binding pocket (41,42) and its polarity (43). The replacement of aspartic acid with cysteine did not lead to significant changes in the spectrum, which points to the presence of an aspartic acid instead of a charged aspartate in the wild type, in agreement with previous findings (23,35).…”

Aureochrome 1 Illuminated: Structural Changes of a Transcription Factor Probed by Molecular Spectroscopy

“…The visible absorption spectrum shows only one broad band in the UVA region, which indicates that the LOV domain of aureochrome1 resembles the LOV2 domain of phototropin [4] and LOV domain of YtvA [36]. This is in line with the fact that T222 and N229 in aureochrome 1, which strongly influence the spectral features in the UVA range [37] are conserved in LOV2 domains. Time-resolved UV/Vis measurements were performed to determine the kinetics of the photocycle intermediates.…”

The Dark Recovery Rate in the Photocycle of the Bacterial Photoreceptor YtvA Is Affected by the Cellular Environment and by Hydration

“…It is therefore important to identify specific amino acids surrounding the chromophore which for the physiological function transfer the chromophore-generated biological signal to the STAS domain, and thus are capable of transducing the interaction with the solvent. We could previously identify several amino acids nearby the chromophore, which upon mutation caused significant effects on the photocycle rate constants [9], [10].…”

“…[10] Proteins in solution used for these experiments were affinity purified from overexpressing E. coli cells employing a His6-tag at their C-terminal end. [10] B. subtilis cells overexpressing YtvA, were generated from an YtvA deletion mutant, transformed with an YtvA-encoding plasmid (U. Krauss, personal communication), and were kindly donated by Ulrich Krauss (FZ Jülich, Germany).…”

“…[9], [10] Moreover, the level of hydration has been shown to affect the latter kinetics in the LOV2 domain of Avena sativa phot1 (referred to as As LOV2). [11] We have thus measured dark recovery kinetics of YtvA in its natural host, B. subtilis , and in E. coli colonies overexpressing YtvA under different humidity level for the wild type protein (wt) and some mutants that had been identified to be instrumental for the dark recovery process.…”