Structure and function in rhodopsin: topology of the C-terminal polypeptide chain in relation to the cytoplasmic loops.

Cysteine mutagenesis and site-directed spin labeling in the C-terminal region of rhodopsin have been used to probe the local structure and proximity of that region to the cytoplasmic loops. Each of the native amino acids in the sequence T335-T340 was replaced with Cys, one at a time. The sulfhydryl groups of all mutants reacted rapidly with the sulfhydryl reagent 4,4'-dithiodipyridine, which indicated a high degree of solvent accessibility. Furthermore, to probe the proximity relationships, a series of double Cys mutants was constructed. One Cys in all sets was at position 338 and the other was at a position in the sequence S240-V250 in the EF interhelical loop, at position 65 in the AB interhelical loop, or at position 140 in the CD interhelical loop. In the dark state, no significant disulfide formation was observed between C338 and C65 or C140 under the conditions used, whereas a relatively rapid disulfide formation was observed between C338 and C242 or C245. Spin labels in the double Cys mutants showed the strongest magnetic interactions between the nitroxides attached to C338 and C245 or C246. Light activation of the double mutant T242C/S338C resulted in slower disulfide formation, whereas interactions between nitroxides at C338 and C245 or C246 decreased. These results suggest the proximity of the C-terminal residue C338 to residues located on the outer face of a cytoplasmic helical extension of the F helix with an apparent increase of distance upon photoactivation.

[1]  J. Bennett.  [36] Paper chromatography and electrophoresis; special procedure for peptide maps , 1967 .

[2]  J. Alderfer,et al.  The first and second cytoplasmic loops of the G-protein receptor, rhodopsin, independently form beta-turns. , 1997, Biochemistry.

[3]  D. Koshland,et al.  Site-directed cross-linking. Establishing the dimeric structure of the aspartate receptor of bacterial chemotaxis. , 1988, The Journal of biological chemistry.

[4]  H. Khorana,et al.  Structural features and light-dependent changes in the cytoplasmic interhelical E-F loop region of rhodopsin: a site-directed spin-labeling study. , 1996, Biochemistry.

[5]  J. Alderfer,et al.  Three-dimensional structure of the cytoplasmic face of the G protein receptor rhodopsin. , 1997, Biochemistry.

[6]  H. Khorana,et al.  Structure and function in rhodopsin. Cysteines 65 and 316 are in proximity in a rhodopsin mutant as indicated by disulfide formation and interactions between attached spin labels. , 1996, Biochemistry.

[7]  H. Khorana,et al.  Mapping light-dependent structural changes in the cytoplasmic loop connecting helices C and D in rhodopsin: a site-directed spin labeling study. , 1995, Biochemistry.

[8]  J. Alderfer,et al.  Structure of the third cytoplasmic loop of bovine rhodopsin. , 1995, Biochemistry.

[9]  L. Salwínski,et al.  A method for distance determination in proteins using a designed metal ion binding site and site-directed spin labeling: evaluation with T4 lysozyme. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[10]  D. Koshland,et al.  Global flexibility in a sensory receptor: a site-directed cross-linking approach. , 1987, Science.

[11]  H. Khorana,et al.  Structure and function in rhodopsin: correct folding and misfolding in two point mutants in the intradiscal domain of rhodopsin identified in retinitis pigmentosa. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[12]  H. Khorana,et al.  Requirement of Rigid-Body Motion of Transmembrane Helices for Light Activation of Rhodopsin , 1996, Science.

[13]  D. Koshland,et al.  Structure of a bacterial sensory receptor. A site-directed sulfhydryl study. , 1988, The Journal of biological chemistry.

[14]  H. Khorana,et al.  Mapping of the amino acids in the cytoplasmic loop connecting helices C and D in rhodopsin. Chemical reactivity in the dark state following single cysteine replacements. , 1995, Biochemistry.

[15]  H. Khorana,et al.  Structure and function in rhodopsin: peptide sequences in the cytoplasmic loops of rhodopsin are intimately involved in interaction with rhodopsin kinase. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[16]  H. Khorana,et al.  Rhodopsin kinase: expression in baculovirus-infected insect cells, and characterization of post-translational modifications. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[17]  K. J. Oh,et al.  Conformation of T4 lysozyme in solution. Hinge-bending motion and the substrate-induced conformational transition studied by site-directed spin labeling. , 1997, Biochemistry.

[18]  H. Khorana,et al.  Glutamic acid-113 serves as the retinylidene Schiff base counterion in bovine rhodopsin. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[19]  J. Javitch,et al.  A cysteine residue in the third membrane-spanning segment of the human D2 dopamine receptor is exposed in the binding-site crevice. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[20]  H. Khorana,et al.  A single amino acid substitution in rhodopsin (lysine 248----leucine) prevents activation of transducin. , 1988, The Journal of biological chemistry.

[21]  H. Khorana,et al.  Formation of the meta II photointermediate is accompanied by conformational changes in the cytoplasmic surface of rhodopsin. , 1993, Biochemistry.

[22]  K. Hideg,et al.  Photoactivated conformational changes in rhodopsin: a time-resolved spin label study. , 1993, Science.

[23]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[24]  J F Murray,et al.  Determination of sulfhydryl groups with 2,2'- or 4,4'-dithiodipyridine. , 1967, Archives of biochemistry and biophysics.

[25]  H. G. Khorana,et al.  Palmitoylation of bovine opsin and its cysteine mutants in COS cells. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[26]  H G Khorana,et al.  Structure and function in rhodopsin. Single cysteine substitution mutants in the cytoplasmic interhelical E-F loop region show position-specific effects in transducin activation. , 1996, Biochemistry.

[27]  S. Kaushal,et al.  Structure and function in rhodopsin: the role of asparagine-linked glycosylation. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[28]  J. Alderfer,et al.  Structure of the carboxy-terminal domain of bovine rhodopsin , 1995, Nature Structural Biology.

[29]  W. Hubbell,et al.  Reactions of the sulfhydryl groups of membrane-bound bovine rhodopsin. , 1978, Membrane biochemistry.

[30]  C. Lai,et al.  Reaction of peptides with fluorescamine on paper after chromatography or electrophoresis. , 1975, Analytical biochemistry.

[31]  H. Khorana,et al.  Expression of a synthetic bovine rhodopsin gene in monkey kidney cells. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[32]  Y. Shin,et al.  Determination of the distance between two spin labels attached to a macromolecule. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[33]  K. Palczewski,et al.  Mechanism of rhodopsin kinase activation. , 1991, The Journal of biological chemistry.

[34]  C. Altenbach,et al.  Investigation of structure and dynamics in membrane proteins using site-directed spin labeling , 1994 .

[35]  H. Khorana,et al.  Structure and Function in Rhodopsin , 1995, The Journal of Biological Chemistry.

[36]  H. Khorana,et al.  Total synthesis of a gene for bovine rhodopsin. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[37]  H. Khorana,et al.  Structure and function in rhodopsin: replacement by alanine of cysteine residues 110 and 187, components of a conserved disulfide bond in rhodopsin, affects the light-activated metarhodopsin II state. , 1994, Proceedings of the National Academy of Sciences of the United States of America.