Signal transduction in light-oxygen-voltage receptors lacking the active-site glutamine.

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.

A Light-Oxygen-Voltage Receptor Integrates Light and Temperature.

Sensory photoreceptors enable organisms to adjust their physiology, behavior, and development in response to light, generally with spatiotemporal acuity and reversibility. These traits underlie the use of photoreceptors as genetically encoded actuators to alter by light the state and properties of heterologous organisms. Subsumed as optogenetics, pertinent approaches enable regulating diverse cellular processes, not least gene expression. Here, we controlled the widely used Tet repressor by coupling to light-oxygen-voltage (LOV) modules that either homodimerize or dissociate under blue light. Repression could thus be elevated or relieved, and consequently protein expression was modulated by light. Strikingly, the homodimeric RsLOV module from Rhodobacter sphaeroides not only dissociated under light but intrinsically reacted to temperature. The limited light responses of wild-type RsLOV at 37 °C were enhanced in two variants that exhibited closely similar photochemistry and structure. One variant improved the weak homodimerization affinity of 40 µM by two-fold and thus also bestowed light sensitivity on a receptor tyrosine kinase. Certain photoreceptors, exemplified by RsLOV, can evidently moonlight as temperature sensors which immediately bears on their application in optogenetics and biotechnology. Properly accounted for, the temperature sensitivity can be leveraged for the construction of signal-responsive cellular circuits.

Photobiologically Directed Assembly of Gold Nanoparticles.

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 unlock these traits for material science, the light-induced homodimerization of light-oxygen-voltage (LOV) photoreceptors is leveraged to control the assembly of gold nanoparticles. Conjugated to genetically encodable LOV proteins, the nanoparticles are monodispersed in darkness but rapidly assemble into large aggregates upon blue-light exposure. The study establishes a new modality for reaction control in macromolecular chemistry and thus augurs enhanced precision in space and time in diverse applications of gold nanoparticles.

Pulsatile illumination for photobiology and optogenetics.

Living organisms exhibit a wide range of intrinsic adaptive responses to incident light. Likewise, in optogenetics, biological systems are tailored to initiate predetermined cellular processes upon light exposure. As genetically encoded, light-gated actuators, sensory photoreceptors are at the heart of these responses in both the natural and engineered scenarios. Upon light absorption, photoreceptors enter a series of generally rapid photochemical reactions leading to population of the light-adapted signaling state of the receptor. Notably, this state persists for a while before thermally reverting to the original dark-adapted resting state. As a corollary, the inactivation of photosensitive biological circuits upon light withdrawal can exhibit substantial inertia. Intermittent illumination of suitable pulse frequency can hence maintain the photoreceptor in its light-adapted state while greatly reducing overall light dose, thereby mitigating adverse side effects. Moreover, several photoreceptor systems may be actuated sequentially with a single light color if they sufficiently differ in their inactivation kinetics. Here, we detail the construction of programmable illumination devices for the rapid and parallelized testing of biological responses to diverse lighting regimes. As the technology is based on open electronics and readily available, inexpensive components, it can be adopted by most laboratories at moderate expenditure. As we exemplify for two use cases, the programmable devices enable the facile interrogation of diverse illumination paradigms and their application in optogenetics and photobiology.