RESUMO
LOV (light oxygen voltage) proteins are photosensors ubiquitous to all domains of life. A variant of the short LOV protein from Dinoroseobacter shibae (DsLOV) exhibits an exceptionally fast photocycle. We performed time-resolved molecular spectroscopy on DsLOV-M49S and characterized the formation of the thio-adduct state with a covalent bond between the reactive cysteine (C72) and C4a of the FMN. By use of a tunable quantum cascade laser, the weak absorption change of the vibrational band of S-H stretching vibration of C57 was resolved with a time resolution of 10 ns. Deprotonation of C72 proceeded with a time constant of 12 µs which tallies the rise of the thio-adduct state. These results provide valuable information for the mechanistic interpretation of light-induced structural changes in LOV domains, which involves the choreographed sequence of proton transfers, changes in electron density distributions, spin alterations of the latter, and transient bond formation and breakage. Such molecular insight will help develop new optogenetic tools based on flavin photoreceptors.
Assuntos
Cisteína/metabolismo , Flavinas/metabolismo , Prótons , Rhodobacteraceae/química , Fatores de Transcrição/metabolismo , Cisteína/química , Flavinas/química , Modelos Moleculares , Estrutura Molecular , Processos Fotoquímicos , Fatores de Tempo , Fatores de Transcrição/químicaRESUMO
The light-oxygen-voltage (LOV) domains of phototropins emerged as essential constituents of light-sensitive proteins, helping initiate blue light-triggered responses. Moreover, these domains have been identified across all kingdoms of life. LOV domains utilize flavin nucleotides as co-factors and undergo structural rearrangements upon exposure to blue light, which activates an effector domain that executes the final output of the photoreaction. LOV domains are versatile photoreceptors that play critical roles in cellular signaling and environmental adaptation; additionally, they can noninvasively sense and control intracellular processes with high spatiotemporal precision, making them ideal candidates for use in optogenetics, where a light signal is linked to a cellular process through a photoreceptor. The ongoing development of LOV-based optogenetic tools, driven by advances in structural biology, spectroscopy, computational methods, and synthetic biology, has the potential to revolutionize the study of biological systems and enable the development of novel therapeutic strategies.
Assuntos
Optogenética , Fototropinas , Oxigênio , Domínios Proteicos/efeitos da radiação , Fototropinas/química , Fototropinas/efeitos da radiação , LuzRESUMO
Fourier-transform infrared (FTIR) spectroscopy using vibrational probes is an ideal tool to detect changes in structure and local environments within biological molecules. However, challenges arise when dealing with weak infrared probes, such as thiocyanates, due to their inherent low signal strengths and overlap with solvent bands. In this protocol we demonstrate:â¢A streamlined approach for the precise extraction of weak infrared absorption lineshapes from a strong solvent background.â¢A protocol combining a spectral filter, background modeling, and subtraction.â¢Our methodology successfully extracts the CN stretching mode peak from methyl thiocyanate at remarkably low concentrations (0.25 mM) in water, previously a challenge for FTIR spectroscopy.This approach offers valuable insights and tools for more accurate FTIR measurements using weak vibrational probes. This enhanced precision can potentially enable new approaches to enhance our understanding of protein structure and dynamics in solution.