1.
Signal transduction in light-oxygen-voltage receptors lacking the adduct-forming cysteine residue.
Abstract:
Light-oxygen-voltage (LOV) receptors sense blue light through the photochemical generation of a covalent adduct between a flavin-nucleotide chromophore and a strictly conserved cysteine residue. Here we show that, after cysteine removal, the circadian-clock LOV-protein Vivid still undergoes light-induced dimerization and signalling because of flavin photoreduction to the neutral semiquinone (NSQ). Similarly, photoreduction of the engineered LOV histidine kinase YF1 to the NSQ modulates activity and downstream effects on gene expression. Signal transduction in both proteins hence hinges on flavin protonation, which is common to both the cysteinyl adduct and the NSQ. This general mechanism is also conserved by natural cysteine-less, LOV-like regulators that respond to chemical or photoreduction of their flavin cofactors. As LOV proteins can react to light even when devoid of the adduct-forming cysteine, modern LOV photoreceptors may have arisen from ancestral redox-active flavoproteins. The ability to tune LOV reactivity through photoreduction may have important implications for LOV mechanism and optogenetic applications.
2.
Biophysical, mutational, and functional investigation of the chromophore-binding pocket of light-oxygen-voltage photoreceptors.
Abstract:
As light-regulated actuators, sensory photoreceptors underpin optogenetics and numerous applications in synthetic biology. Protein engineering has been applied to fine-tune the properties of photoreceptors and to generate novel actuators. For the blue-light-sensitive light-oxygen-voltage (LOV) photoreceptors, mutations near the flavin chromophore modulate response kinetics and the effective light responsiveness. To probe for potential, inadvertent effects on receptor activity, we introduced these mutations into the engineered LOV photoreceptor YF1 and determined their impact on light regulation. While several mutations severely impaired the dynamic range of the receptor (e.g., I39V, R63K, and N94A), residue substitutions in a second group were benign with little effect on regulation (e.g., V28T, N37C, and L82I). Electron paramagnetic resonance and absorption spectroscopy identified correlated effects for certain of the latter mutations on chromophore environment and response kinetics in YF1 and the LOV2 domain from Avena sativa phototropin 1. Carefully chosen mutations provide a powerful means to adjust the light-response function of photoreceptors as demanded for diverse applications.
