Studies on the stability of the human cone visual pigments.
The retina of vertebrates contains two kinds of photoreceptor cells, rods and cones, which contain their specific visual pigments that are responsible for scotopic and photopic vision, respectively. In cone photoreceptor cells, there are three types of color pigments: blue, green and red, each with a distinctive absorption maximum. The goal of this investigation was to identify optimal conditions under which these pigments could be obtained and isolated in a stable form, thereby facilitating structural studies using high-resolution approaches. For this purpose, all three human cone opsins were initially expressed in mammalian cells, reconstituted with 11-cis retinal, detergent solubilized, purified and their stability compared with rod rhodopsin. As all three pigments showed dramatically reduced stability relative to rhodopsin, site-directed mutagenesis was used in an attempt to engineer stability into the green cone pigment. The mutations introduced some structural motifs and sites of posttranslational modification present in rhodopsin, as well as amino acid substitutions that have been found to stabilize the rod opsin apo-protein. We also modified the hydrophobic environment of the green cone pigment by varying the detergent and detergent/lipid composition used during solubilization and purification, and compared them with the retinal reconstituted pigment in membranes. Our results show that these changes do not significantly improve the inherent instability of the human cone pigments, and in some cases, lead to a decrease in stability and protein aggregation. We conclude that further efforts are required to stabilize the human cone pigments in a form suitable for high-resolution structural studies.
Ramon
Eva
E
Department of Biochemistry and Molecular Biology, University of Texas Health Science Center-Houston, Houston, TX, USA.
Mao
Xiang
X
Ridge
Kevin D
KD
D000595:Amino Acid Sequence; D002460:Cell Line; D006801:Humans; D008055:Lipids / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D055355:Opsins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017949:Retinal Cone Photoreceptor Cells / Q000737:chemistry* / Q000378:metabolism*; D012168:Retinal Pigments / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D013053:Spectrophotometry
D008055:Lipids; D055355:Opsins; D012168:Retinal Pigments