Mechanism-Guided Reengineering of Light-Oxygen-Voltage Photoreceptors

• Applicant: Professor Dr. Andreas Möglich
• Subject Area: Biochemistry
• Term: since 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 420423318

Sensory photoreceptors underlie manifold adaptive responses to light across diverse organisms. Photoreceptors double as genetically encodable actuators in optogenetics and enable the acute and reversible light-dependent control of various cellular parameters and processes. As one class, light-oxygen-voltage (LOV) receptors bind flavin nucleotides as chromophores to detect blue light. Photon absorption ushers in the reversible LOV photocycle and yields the signaling state characterized by a covalent bond between the flavin and a nearby conserved cysteine residue. Formation of this thioadduct entails a formal reduction of the flavin and concomitant protonation at its N5 atom, a key event both necessary and sufficient for downstream signal transduction. In this project, we pursue the mechanistic characterization of LOV receptors incapable of forming a stable cysteinyl adduct which are adept at converting light stimuli into diverse output modes. We will elucidate how the flavin reduction midpoint potential can be altered at will and how pertinent alterations affect photochemical responses. Fluorescent LOV modules with stratified redox sensitivities will serve as reporters suitable for addressing which reduction state LOV receptors adopt inside cells. Enhanced fluorescence in LOV domains is commonly achieved by removal of the active-site cysteine which however simultaneously incurs higher propensity for sensitizing oxygen. Our efforts are hence directed at devising LOV derivatives with strong fluorescence yet minimal side reactivity. A final project branch capitalizes on our recent discovery of a new type of irreversible photoadduct that elicits downstream signal transduction in LOV receptors. We will investigate how the irreversible photoadduct can be installed in conventional LOV receptors, and how it can be leveraged for optogenetics. LOV receptors exhibiting the irreversible adduct exist in nature and constitute a previously undocumented, rare photoreceptor clade. Both engineered and natural occurring photoreceptors forming the irreversible photoadduct represent attractive tools for biotechnological application and powerful paradigms for mechanistic investigation.

• DFG Programme: Research Grants