Serine 85 in Transmembrane Helix 2 of Short-Wavelength Visual Pigments Interacts with the Retinylidene Schiff Base Counterion ² Abhiram Dukkipati, ‡,§ Bryan W. Vought, ‡,| Deepak Singh, ‡,⊥ Robert R. Birge,* ,‡,# and Barry E. Knox* ,§ Departments of Biochemistry and Molecular Biology and of Ophthalmology, SUNY Upstate Medical UniVersity, 750 East Adams Street, Syracuse, New York 13210, and Departments of Chemistry and Biology, Syracuse UniVersity, 111 College Place, Syracuse, New York 13244-4100 ReceiVed June 27, 2001; ReVised Manuscript ReceiVed October 10, 2001 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, λ 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. Vertebrate vision is mediated by five distinct families of visual pigments (1). These pigments are prototypical hep- tahelical GPCRs, 1 with an 11-cis-retinal chromophore co- valently bound via a Schiff base to a lysine in transmembrane helix 7 of the opsin apoprotein (2, 3). Absorption of a photon isomerizes 11-cis-retinal to all-trans-retinal, resulting in a series of conformational changes in the protein (4, 5). The final active conformation, meta II, activates the G protein, transducin, initiating the downstream signaling cascade (6). Unlike other GPCRs, in which the ligand acts as an agonist, the covalently bound retinal acts as an antagonist and prevents the receptor from activating the signaling cascade in the dark. There are two fundamentally different realms of photo- receptor function: dim light (scotopic) typically taking place in rods and photopic illuminance taking place in cones (7). In response to the wide-ranging lighting conditions in which cones and rods function, markedly different response char- acteristics have developed (8). Cone responses adapt rapidly to changes in light intensity and are noisy but do not saturate to ambient light levels. Additionally, cones have adapted to respond to a wide spectral range of light, from the ultraviolet to far-red. Cone visual pigments are responsible for spectral sensitivities of cones and may play an important role in determining the response dynamics of the cell. Cone visual pigments have a number of properties that distinguish them from the rhodopsins: a shorter lifetime of the active protein conformation compared to rhodopsins and rapid recovery postbleaching. Moreover, the retinal binding pocket is more solvent accessible in the dark, demonstrated by hydroxy- lamine-induced bleaching. The molecular features that determine the differences between rod and cone pigment properties have not been well characterized. Numerous mutations that alter the spectral tuning and several that change the photobleaching pathway in a variety of rod and cone opsins have not yet clarified these properties (1, 9-14). The recent elucidation of the structure of bovine rhodopsin to 2.8 Å (15) opens the way for crucial insights into the ² This work was supported in part by NIH Grants GM-34548 (to R.R.B.) and EY-12975 and EY-11256 (to B.E.K.), the W. M. Keck Center for Molecular Electronics at Syracuse University, and a grant from the Research to Prevent Blindness Foundation. * Correspondence should be addressed to either B.E.K. [tel, (315) 464-8719; fax, (315) 464-8750; e-mail, knoxb@mail.upstate.edu] or R.R.B. [tel, (860) 486-6720; fax, (860) 486-2981; e-mail, rbirge@ uconn.edu]. ‡ Syracuse University. § SUNY Upstate Medical University. | Present address: Department of Biological Chemistry and Molec- ular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. ⊥ Present address: GeneFormatics Inc., 5830 Oberlin Drive, Suite 200, San Diego, CA 92121. # Present address: Departments of Chemistry and Molecular and Cell Biology, University of Connecticut, 55 North Eagleville Road, Storrs, CT 06269. 1 Abbreviations: GPCR, G protein coupled receptor; VCOP, violet cone opsin; TM no., transmembrane helix no.; SWS, short-wavelength sensitive; RH, rhodopsins; M/LWS, medium/long wavelength sensitive; nm, nanometers; PSS(xyz), photostationary state generated by illumina- tion at wavelength xyz in nanometers; B1, photostationary state 395 formed at 45 K; B2, photostationary state 395 formed at 75 K; DM, N-dodecyl -D-maltoside; ROS, rod outer segment; batho and lumi, discrete thermal intermediates of the visual opsin bleaching pathway. 15098 Biochemistry 2001, 40, 15098-15108 10.1021/bi011354l CCC: $20.00 © 2001 American Chemical Society Published on Web 11/21/2001