| Retinylidene proteins, consisting of type I and type II opsins, are integral membrane proteins that perform light-dependent functions. Two structural features are conserved in all retinylidene proteins: the G protein coupled receptor fold and an active-site lysine located in transmembrane helix seven. Determining the evolutionary relationship between type I and type II opsins has proved to be a challenge because there is no significant sequence similarity between the two types. One hypothesis is that random mutations accumulated over one billion years of evolution have obscured sequence similarity that could provide evidence of homology. A competing hypothesis is that the two types of opsins arose through convergent evolution. In this work, I test for a functional constraint, the major tenet of convergent evolution.;To test for a functional constraint, I designed mutants that relocate the conserved active-site lysine to different secondary structure elements in the visual pigment bovine rhodopsin, the canonical type II opsin. I found that the lysine can be relocated to three positions while maintaining the ability to form a pigment with 11-cis-retinal and activate transducin in a light-dependent manner. I also tested whether the active-site lysine position is fixed in modern proteins, by analyzing potential evolutionary intermediates with two lysines in the active site. All four of these mutants bind 11- cis-retinal and activate transducin in a light dependent manner. Finally, from an alignment of 489 type II opsins I identified two sets of potential evolutionary intermediates, each containing two lysines in the active site. These results indicate there is no functional constraint requiring the active-site lysine to be in helix seven, undermining the convergent evolution hypothesis. Furthermore, it appears evolution could relocate the active-site lysine in modern proteins, via an intermediate with two active-site lysines. |
