Physical methods for studying flavoprotein photoreceptors

Abstract

Molecular mechanisms of dark-to-light state transitions in flavoprotein photoreceptors have been the subject of intense investigation. Blue-light sensing flavoproteins fall into three general classes that share aspects of their activation processes: LOV domains, BLUF proteins and cryptochromes. In all cases, light-induced changes in flavin redox, protonation and bonding states result in hydrogen-bond and conformational rearrangements important for regulation of downstream targets. Physical characterization of these flavoprotein states can provide valuable insights into biological function, but clear conclusions are often not trivial to obtain owing to challenges in data collection and interpretation. In this chapter, we briefly review the three classes of flavoprotein photoreceptors and provide basic methods for their recombinant production, reconstitution with flavin cofactor, and characterization. We then relate best practices and special considerations for applying several types of spectroscopies, redox potential measurements, and X-ray scattering experiments to this family of flavoprotein. The methods presented are generally accessible to most laboratories.

Residue interaction dynamics in Vaucheria aureochrome1 light‐oxygen‐voltage: Bridging theory and experiments

“…A small peak around 415 nm though matches neutral hydroquinone state of flavin in time‐dependent density functional theory calculations, 28 presence of doublet at 360 and 378 nm makes the spectrum difficult to explain. However, the spectral characteristics in the visible region do not completely match with neutral hydroquinone state of protein‐bound flavin 31 . Given our protocols for protein purification, it is rather unlikely that free flavin could contaminate the protein solution.…”

“…However, the spectral characteristics in the visible region do not completely match with neutral hydroquinone state of protein-bound flavin. 31 Given our protocols for protein purification, it is rather unlikely that free flavin could contaminate the protein solution. While further investigation is necessary to explain the small peak around 415 nm (also in L284A-F298A), Kasahara et al associate similar characteristics in double Arg mutant of LOV/LOV protein with the loss of FMN binding.…”

Enhancing the performance of Magnets photosensors through directed evolution

“…T83M at the entrance of the water channel harboring the flavin chromophore and K119N in the FAD loop (Supplementary Figure 25 E) were both characterized as single mutations, and might alter chromophore binding and positioning, again allowing for cysteine-flavin adduct formation at lower light intensities. To gain more definite answers, x-ray crystallography and UV-vis spectroscopy 55 could shed more light on the underlying structural and mechanistic functions of these amino acid exchanges. This is out of the scope of our engineering-driven approach and should be investigated in future studies.…”

Flavin Charge Redistribution upon Optical Excitation Is Independent of Solvent Polarity

“…Flavin cofactors have recently been found to couple endergonic and exergonic reduction of separate substrates via electron bifurcation and participate in a plethora of covalent catalytic mechanisms . Beyond catalysis, flavin cofactors mediate blue-light photoreception in blue-light sensing using FAD (BLUF) domains, light-oxygen-voltage (LOV) domains, cryptochromes, and the intermediate CRY-DASH, which exhibits both photoreception and limited DNA photolyase functionalities. One reason that flavin cofactors are so ubiquitous is that their ground- and excited-state reduction potentials, already usefully poised for exchanging electrons among common metabolites, are electrostatically tunable by selective perturbation from the flavoprotein active site’s charged and polar residues. − The ground-state two-electron reduction potential of flavin at physiological pH is continuously tunable through a range of over 500 mV .…”