Physical methods for studying flavoprotein photoreceptors

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 challenging to draw owing to complexities of data collection and interpretation. In this chapter, we briefly review the three classes of flavoprotein photoreceptors and provide methods for their recombinant production, reconstitution with flavin cofactor, and characterization. We then relate best practices and special considerations for the application of several types of spectroscopies, redox potential measurements, and X-ray scattering experiments to photosensitive flavoproteins. The methods presented are generally accessible to most laboratories.