Additive and epistatic effects influence spectral tuning in molluscan retinochrome opsin

Abstract

The relationship between genotype and phenotype is nontrivial due to often complex molecular pathways that make it difficult to unambiguously relate phenotypes to specific genotypes. Photopigments, an opsin apoprotein bound to a light-absorbing chromophore, present an opportunity to directly relate the amino acid sequence to an absorbance peak phenotype ({lambda}max). We examined this relationship by conducting a series of site-directed mutagenesis experiments of retinochrome, a non-visual opsin, from two closely related species: the common bay scallop, Argopecten irradians, and the king scallop, Pecten maximus. Using protein folding models, we identified three amino acid sites of likely functional importance and expressed mutated retinochrome proteins in vitro. Our results show that the mutation of amino acids lining the opsin binding pocket are responsible for fine spectral tuning, or small changes in the {lambda}max of these light sensitive proteins Most mutations caused a blue shift regardless of the retinochrome background, with shifts ranging from a 12 nm blue shift to a 5 nm red shift from the wild-type {lambda}max. These mutations do not show an additive effect, but rather suggests the presence of epistatic interactions. This work highlights the importance of binding pocket shape in the evolution of spectral tuning and builds on our ability to relate genotypic changes to phenotypes in an emerging model for opsin functional analysis. Author summarySite-directed mutagenesis determined that spectral tuning in retinochrome is not solely additive, but is influenced by intra-molecular epistasis.