Signal Transduction in Light-Oxygen-Voltage Receptors Lacking the Active-Site Glutamine

Abstract

In nature as in biotechnology, light-oxygen-voltage (LOV) photoreceptors perceive blue light to elicit spatiotemporally defined cellular responses. Photon absorption drives thioadduct formation between a conserved cysteine and the flavin chromophore. An equally conserved, proximal glutamine processes the resultant flavin protonation into downstream hydrogen-bond rearrangements. Here, we report that this glutamine, long deemed essential, is generally dispensable. In its absence, several LOV receptors invariably retained productive, if often attenuated, signaling responses. Structures of a LOV paradigm at around 1 Å resolution revealed highly similar light-induced conformational changes, irrespective of whether the glutamine is present. Naturally occurring, glutamine-deficient LOV receptors likely serve as bona fide photoreceptors, as we showcase for a diguanylate cyclase. We propose that without the glutamine, water molecules transiently approach the chromophore and thus propagate flavin protonation downstream. Signaling without glutamine appears intrinsic to LOV receptors, which pertains to biotechnological applications and suggests evolutionary descendance from redox-active flavoproteins.

Fine spectral tuning of a flavin-binding fluorescent protein for multicolor imaging

“…Previously, several attempts have been made to generate flavin-based fluorescent proteins with shifted spectra, with moderate success (33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43). In this work, we mutated a previously untargeted amino acid position, that of photoactive cysteine (corresponding to A85 in CagFbFP), in addition to mutations of other residues near the flavin (I52 and Q148, Figure 3A), and identified a palette of 22 variants that uniformly cover the range of emission spectra with the maxima from 486 to 512 nm (Figure 3C).…”

“…Most of the mutations described to date lead to minor hypsochromic shifts, with the exception of those in the singlet oxygen generator miniSOG2 protein (33). Replacement of the conserved glutamine adjacent to FMN with a valine or a leucine results in a blue shift of ~10 nm (34)(35)(36)(37), although a similar mutation in the phiSOG has almost no effect on the spectrum (36). Replacement of the glutamine with positively charged amino acids, contrary to the prediction (38), also resulted in hypsochromic shifts (39,40).…”

Optogenetic Control of Bacterial Expression by Red Light

“…23 While in darkness, YF1 acts as a net kinase on FixJ, blue light converts it into a net phosphatase that efficiently removes phosphoryl groups from phospho-FixJ. 23,24 Expression of target genes is thus lowered by around 15-fold under blue light compared to that in darkness. The pDawn plasmid derives from pDusk and harbors a gene-inversion cassette based on the λ phage cI repressor; blue light hence leads to an upregulation of target gene expression by several 100-fold.…”