title: Serine 85 in transmembrane helix 2 of short-wavelength visual pigments interacts with the retinylidene Schiff base counterion.
abstract: Short-wavelength cone visual pigments (SWS1) are responsible for detecting light from 350 to 430 nm. Models of this class of pigment suggest that TM2 has extensive contacts with the retinal binding pocket and stabilizes interhelical interactions. The role of TM2 in the structure-function of the Xenopus SWS1 (VCOP, lambda(max) = 427 nm) pigment was studied by replacement of the helix with that of bovine rhodopsin and also by mutagenesis of highly conserved residues. The TM2 chimera and G78D, F79L, M81E, P88T, V89S, and F90V mutants did not produce any significant spectral shift of the dark state or their primary photointermediate formed upon illumination at cryogenic temperatures. The mutant G77R (responsible for human tritanopia) was completely defective in folding, while C82A and F87T bound retinal at reduced levels. The position S85 was crucial for obtaining the appropriate spectroscopic properties of VCOP. S85A and S85T did not bind retinal. S85D bound retinal and had a wild-type dark state at room temperature and a red-shifted dark state at 45 K and formed an altered primary photointermediate. S85C absorbed maximally at 390 nm at neutral pH and at 365 nm at pH >7.5. The S85C dark state was red shifted by 20 nm at 45 K and formed an altered primary photointermediate. These data suggest that S85 is involved in a hydrogen bond with the protonated retinylidene Schiff base counterion in both the dark state and the primary photointermediate.
authors.affiliation: Department of Biochemistry, SUNY Upstate Medical University, Syracuse, New York 13210, USA.
authors.forename: A
authors.initials: A
authors.lastname: Dukkipati
authors.forename: B W
authors.initials: BW
authors.lastname: Vought
authors.forename: D
authors.initials: D
authors.lastname: Singh
authors.forename: R R
authors.initials: RR
authors.lastname: Birge
authors.forename: B E
authors.initials: BE
authors.lastname: Knox
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D006801:Humans; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010777:Photochemistry; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017299:Rod Opsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012545:Schiff Bases; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D013053:Spectrophotometry
chemical_list: D011993:Recombinant Fusion Proteins; D017299:Rod Opsins; D012545:Schiff Bases; C095853:short-wavelength opsin; D012694:Serine