Exploring Structural and Functional Variation Among Opsin Classes

Opsins comprise a family of proteins belonging to the superfamily of G-protein coupled receptors (GPCRs). The opsins combined with vitamin-A derived chromophores make up the photopigments responsible for such diverse functions as image- and nonimage-forming vision, entrainment of circadian cycles, and the pupillary light response. The elucidation of the structural and biochemical properties of photopigments is of extreme importance to understanding the biological processes that they control. Currently the crystal structure of two photopigments have been solved; bovine rhodopsin from the vertebrate visual opsins, and squid rhodopsin from the invertebrate visual opsins. In the vertebrate visual opsins, five main subclasses exist; the RH1 class containing bovine rhodopsin makes up the rod based opsins, and the SWS1, SWS2, RH2, and M/LWS classes make up the cone based opsins. I question how similar structurally and biochemically the cone opsin classes are to the RH1 class. Using in vitro G-protein activation assays it is shown that mutations targeting key structural bonds within the cone opsin classes, compared to the RH1 class, result in different profiles of biochemical behavior reflective of structural variation surrounding the chromophore-binding pocket. This structural variation is further explored through the use of spin-labeling techniques aimed at analyzing the helical arrangement within the M/LWS cone opsin class compared to the RH1 class. In addition, a sixth class of opsins, the melanopsins, which maintains greater sequence identity to the invertebrate visual opsins than to the vertebrate visual opsins, is analyzed to identify the location of the counterion to the protonated Schiff base. The counterion is a key functional residue within the opsin family responsible for stabilizing the inactive dark state pigment as well as tuning the wavelength absorbance into the visible spectrum. Through spectroscopic analysis of various melanopsin mutants, evidence is presented that shows the counterion residue is not conserved across opsin groups. Through the evolution of the opsins, the location of the counterion has changed at least twice. This work highlights the large level of complexity that exists in the structure of the opsin family emphasizing the need for further structural analysis