In nature as in biotechnology, light-oxygen-voltage 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 light-oxygen-voltage receptors invariably retained productive, if often attenuated, signaling responses. Structures of a light-oxygen-voltage paradigm at around 1 Å resolution revealed highly similar light-induced conformational changes, irrespective of whether the glutamine is present. Naturally occurring, glutamine-deficient light-oxygen-voltage 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 light-oxygen-voltage receptors, which pertains to biotechnological applications and suggests evolutionary descendance from redox-active flavoproteins.

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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.

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receptors (Figure 1C). Nanoparticles in general are of fundamental interest for a variety of industrial processes. [3,4] AuNPs in specific underpin diverse applications in, e.g., biological sensing and imaging, [5][6][7] drug delivery, [8] and intracellular gene regulation. [9] Moreover, AuNPs of various shapes and sizes were ordered and arranged in defined architectures. [5][6][7][8][9][10][11][12] Nanoparticle assembly was for instance controlled by various ligands, [13][14][15] but also by external stimuli, such as magnetic fields or light. [16][17][18][19][20][21][22][23][24] These advances notwithstanding, the controlled higherorder assembly in response to external cues remains challenging, but worthwhile to explore because it could significantly extend the application scope of AuNPs. In particular, the interfacing of the inorganic nanoparticles with genetically encodable, adaptable, light-responsive proteins augurs innovative use cases. As a cue signal, light appears ideal as it can be applied non-invasively and supports high spatial and temporal precision, which is not least evidenced by ample applications in biology and biotechnology. [2] To achieve LOV-directed AuNP assembly, we envisioned a direct link between the organic photoreceptor proteins and the inorganic particles. Initial attempts to covalently couple the light-responsive proteins to citrate-capped AuNPs via gold sulfide bonds failed, because unspecific interactions (owing to cysteine residues within the protein and to the presence of salt ions, required for protein stability) invariably triggered nanoparticle aggregation and precipitation. [25,26] Therefore, we opted for the non-covalent attachment of the LOV receptors through nickel coordination chemistry. This strategy necessitates that the photoreceptors be equipped with hexa-histidine (His 6 ) tags and the AuNPs be functionalized with Ni 2+ -nitrilotriacetic acid (NTA) ligands (Figure 1C). [10,21,27] We selected two wellcharacterized LOV domains from Phaeodactylum tricornutum aureochrome 1a (Ptaur) and from Neurospora crassa Vivid (NcVVD), both previously shown to undergo light-induced homodimerization [28,29] and used to bestow light sensitivity on cellular processes. [2] Size-exclusion chromatography (SEC) confirmed that in their unmodified forms both LOV proteins adopt homogenous states in either darkness or blue light (Figure S1, Supporting Information); the apparent molecular size under blue light was higher, indicative of light-induced dimerization. Next, we introduced His 6 tags at either the N or C termini of the LOV domains, separated by short glycine-serine linkers. N-terminal modification of Ptaur led to constitutive dimerization independent of light and accompanied by aggregation, whereas C-terminal modification incurred protein insolubility. The sensitivity of the Ptaur N-and C-terminal segments likely In nature, photoreceptor proteins undergo molecular responses to light, that exhibit supreme fidelity in time and space and generally occur under mild reaction conditions. To un...