Abstract
Light Oxygen Voltage (LOV) proteins are widely used in optogenetic devices, however universal signal transduction pathways and photocycle mechanisms remain elusive. In particular, short-LOV (sLOV) proteins have been discovered in bacteria and fungi, containing only the photoresponsive LOV element without any obvious signal transduction domains. These sLOV proteins may be ideal models for LOV domain function due to their ease of study as full-length proteins. Unfortunately, characterization of such proteins remains limited to select systems. Herein, we identify a family of bacterial sLOV proteins present in Methylocystis. Sequence analysis of Methylocystis LOV proteins (McLOV) demonstrates conservation with sLOV proteins from fungal systems that employ competitive dimerization as a signaling mechanism. Cloning and characterization of McLOV proteins confirms functional dimer formation and reveal unexpected photocycle mechanisms. Specifically, some McLOV photocycles are insensitive to external bases such as imidazole, in contrast to previously characterized LOV proteins. Mutational analysis identifies a key residue that imparts insensitivity to imidazole in two McLOV homologs and affects adduct decay by two orders of magnitude. The resultant data identifies a new family of LOV proteins that indicate a universal photocycle mechanism may not be present in LOV proteins.
Highlights
Light Oxygen Voltage (LOV) domain containing proteins enable organisms to adapt to alterations in environmental stimuli including blue-light, oxygen sensing, and stress responses
To search for similar signal transduction pathways in bacterial species we searched for sLOV proteins with high homology to ENVOY (ENV1) from Trichoderma reesei
Genome analysis revealed that Methylocystis bacteria contain two LOV proteins with moderate homology to ENV1, including the presence of a key Cysteine residue required for signal transduction in fungal sLOV proteins (Fig 1A and 1B) [25]
Summary
Light Oxygen Voltage (LOV) domain containing proteins enable organisms to adapt to alterations in environmental stimuli including blue-light, oxygen sensing, and stress responses. LOV proteins are typically modular in nature, where the stimuli-responsive LOV domain is coupled to N- or C-terminal signal transduction elements [1,2,3]. The photoactive LOV domain couples changes in environmental variables to affect small molecule chemistry. In LOV domains this involves the absorption of blue-light by a flavin (FMN or FAD) cofactor to form a covalent adduct with a conserved Cysteine residue. Blue-light induced formation of the flavin C4a adduct enables allosteric regulation of signal transduction elements to regulate diverse biological function [4].
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