In Vivo and In Vitro Characterization of Cyclase and Phosphodiesterase Rhodopsins.

Rhodopsins with enzymatic activity were found in microbes, in 2004 hypothetically from sequence data and since 2014 by experimental proof. So far three different types are known: light-activated guanylyl cyclase opsins (Cyclop) in fungi, light-inhibited two-component guanylyl cyclase opsins (2c-Cyclop) in green algae, and rhodopsin phosphodiesterases (RhoPDE) in choanoflagellates. They are integral membrane proteins with eight transmembrane helices (TM), different to the other microbial (type I) rhodopsins with 7 TM. Therefore, we propose a classification as type Ib rhodopsins for opsins with 8 TM and type Ia for the ones with 7 TM. To characterize those rhodopsins or their mutants, the expression in Xenopus laevis oocytes proved to be an efficient strategy. Functional analysis was initially performed "in oocyte" (in vivo), but more detailed characterization can be obtained with an in vitro assay. In this chapter, we describe procedures how to extract membranes from oocytes after cRNA microinjection and heterologous protein expression. Enzymatic activity of these membranes is then analyzed under different illumination conditions. In addition, fluorescent labeling of the rhodopsins is employed to quantify the expression level and the absolute activity of designed mutants. We discuss strengths and pitfalls, associated with this expression system, and strategies for selecting potentially useful optogenetic tools.