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PMID: 11106502 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

Light-induced conformational changes of rhodopsin probed by fluorescent alexa594 immobilized on the cytoplasmic surface.

Biochemistry ·Vol. 39 ·No. 49 ·2000-12-12 ·Pages 15225-33

Imamoto Y, Kataoka M, Tokunaga F, Palczewski K

Abstract

A novel fluorescence method has been developed for detecting the light-induced conformational changes of rhodopsin and for monitoring the interaction between photolyzed rhodopsin and G-protein or arrestin. Rhodopsin in native membranes was selectively modified with fluorescent Alexa594-maleimide at the Cys(316) position, with a large excess of the reagent Cys(140) that was also derivatized. Modification with Alexa594 allowed the monitoring of fluorescence changes at a red excitation light wavelength of 605 nm, thus avoiding significant rhodopsin bleaching. Upon absorption of a photon by rhodopsin, the fluorescence intensity increased as much as 20% at acidic pH with an apparent pK(a) of approximately 6.8 at 4 degrees C, and was sensitive to the presence of hydroxylamine. These findings indicated that the increase in fluorescence is specific for metarhodopsin II. In the presence of transducin, a significant increase in fluorescence was observed. This increase of fluorescence emission intensity was reduced by addition of GTP, in agreement with the fact that transducin enhances the formation of metarhodopsin II. Under conditions that favored the formation of a metarhodopsin II-Alexa594 complex, transducin slightly decreased the fluorescence. In the presence of arrestin, under conditions that favored the formation of metarhodopsin I or II, a phosphorylated, photolyzed rhodopsin-Alexa594 complex only slightly decreased the fluorescence intensity, suggesting that the cytoplasmic surface structure of metarhodopsin II is different in the complex with arrestin and transducin. These results demonstrate the application of Alexa594-modified rhodopsin (Alexa594-rhodopsin) to continuously monitor the conformational changes in rhodopsin during light-induced transformations and its interactions with other proteins.

MeSH Terms
Amino Acid Sequence Animals Arrestin/metabolism Binding, Competitive Cattle Cell Membrane Cysteine/chemistry Fluorescent Dyes Hydrogen-Ion Concentration Light Models, Molecular Molecular Sequence Data Organic Chemicals Phosphorylation Photolysis Protein Binding Protein Conformation/radiation effects Protein Structure, Secondary Rhodopsin/chemistry,radiation effects Temperature Transducin/metabolism
Chemicals
Alexa594 Arrestin Fluorescent Dyes Organic Chemicals Rhodopsin Transducin Cysteine
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Imamoto Y
Graduate School of Materials Science, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan. imamoto@ms.aist-nara.ac.jp
Kataoka M
Tokunaga F
Palczewski K
References (57)
57 references, click to expand
  1. The influence of arrestin (48K protein) and rhodopsin kinase on visual transduction.
    Neuron. 1992 Jan;8(1):117-26 PMID: 1309646
  2. Light- and GTP-regulated interaction of GTPase and other proteins with bovine photoreceptor membranes.
    Nature. 1980 Feb 7;283(5747):587-9 PMID: 6101903
  3. Micropatterned immobilization of a G protein-coupled receptor and direct detection of G protein activation.
    Nat Biotechnol. 1999 Nov;17(11):1105-8 PMID: 10545918
  4. Circular dichroism of metaiodopsin II and its binding to transducin: a comparative study between meta II intermediates of iodopsin and rhodopsin.
    Biochemistry. 1994 Apr 26;33(16):4940-6 PMID: 8161555
  5. State-dependent disulfide cross-linking in rhodopsin.
    Biochemistry. 1999 Sep 14;38(37):12028-32 PMID: 10508406
  6. Plasmon resonance spectroscopy: probing molecular interactions within membranes.
    Trends Biochem Sci. 1999 Jun;24(6):213-9 PMID: 10366845
  7. Interplay between hydroxylamine, metarhodopsin II and GTP-binding protein in bovine photoreceptor membranes.
    Biochim Biophys Acta. 1983 Oct 31;725(1):60-70 PMID: 6313051
  8. Formation of the meta II photointermediate is accompanied by conformational changes in the cytoplasmic surface of rhodopsin.
    Biochemistry. 1993 Nov 16;32(45):12025-32 PMID: 8218279
  9. TAUTOMERIC FORMS OF METARHODOPSIN.
    J Gen Physiol. 1963 Nov;47:215-40 PMID: 14080814
  10. Difference in molecular properties between chicken green and rhodopsin as related to the functional difference between cone and rod photoreceptor cells.
    Biochemistry. 1995 Aug 22;34(33):10525-31 PMID: 7654707
  11. Effects of carboxyl-terminal truncation on the stability and G protein-coupling activity of bovine rhodopsin.
    Biochemistry. 1994 Jun 21;33(24):7587-93 PMID: 8011624
  12. Light-regulated binding of rhodopsin kinase and other proteins to cattle photoreceptor membranes.
    Biochemistry. 1978 Oct 17;17(21):4389-95 PMID: 718845
  13. Signaling states of rhodopsin. Retinal provides a scaffold for activating proton transfer switches.
    J Biol Chem. 2000 Jun 30;275(26):19713-8 PMID: 10770924
  14. Two different forms of metarhodopsin II: Schiff base deprotonation precedes proton uptake and signaling state.
    Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7849-53 PMID: 8356093
  15. The role of sulfhydryl groups in the bleaching and synthesis of rhodopsin.
    J Gen Physiol. 1952 May;35(5):797-821 PMID: 14955620
  16. Phosphorylation modulates the affinity of light-activated rhodopsin for G protein and arrestin.
    Biochemistry. 2000 May 16;39(19):5738-49 PMID: 10801324
  17. Molecular basis of visual excitation.
    Science. 1968 Oct 11;162(3850):230-9 PMID: 4877437
  18. Mutation of the fourth cytoplasmic loop of rhodopsin affects binding of transducin and peptides derived from the carboxyl-terminal sequences of transducin alpha and gamma subunits.
    J Biol Chem. 2000 Jan 21;275(3):1937-43 PMID: 10636895
  19. Structure and function in rhodopsin: covalent crosslinking of the rhodopsin (metarhodopsin II)-transducin complex--the rhodopsin cytoplasmic face links to the transducin alpha subunit.
    Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7643-7 PMID: 8052635
  20. Two adjacent cysteine residues in the C-terminal cytoplasmic fragment of bovine rhodopsin are palmitylated.
    FEBS Lett. 1988 Mar 28;230(1-2):1-5 PMID: 3350146
  21. Pre-lumirhodopsin and the bleaching of visual pigments.
    Nature. 1963 Mar 30;197:1279-86 PMID: 14002749
  22. Kinetics, binding constant, and activation energy of the 48-kDa protein-rhodopsin complex by extra-metarhodopsin II.
    Biochemistry. 1989 Feb 21;28(4):1770-5 PMID: 2719933
  23. Evidence for differently protonated forms of metarhodopsin II as intermediates in the decay of membrane-bound cattle rhodopsin.
    Biochem Biophys Res Commun. 1978 Jul 28;83(2):457-65 PMID: 29625
  24. Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates.
    J Histochem Cytochem. 1999 Sep;47(9):1179-88 PMID: 10449539
  25. Rhodopsin and bacteriorhodopsin: structure-function relationships.
    FEBS Lett. 1982 Nov 8;148(2):179-91 PMID: 6759163
  26. Three cytoplasmic loops of rhodopsin interact with transducin.
    Proc Natl Acad Sci U S A. 1989 Sep;86(18):6878-82 PMID: 2780545
  27. Visual pigment: G-protein-coupled receptor for light signals.
    Cell Mol Life Sci. 1998 Dec;54(12):1299-315 PMID: 9893707
  28. Comparative study on the chromophore binding sites of rod and red-sensitive cone visual pigments by use of synthetic retinal isomers and analogues.
    Biochemistry. 1990 Mar 27;29(12):3133-40 PMID: 2140051
  29. Light-induced interaction between rhodopsin and the GTP-binding protein. Metarhodopsin II is the major photoproduct involved.
    Eur J Biochem. 1982 Sep;127(1):97-103 PMID: 6291939
  30. Texas Red, a hydrophilic, red-emitting fluorophore for use with fluorescein in dual parameter flow microfluorometric and fluorescence microscopic studies.
    J Immunol Methods. 1982;50(2):193-204 PMID: 6806389
  31. Protonation states of membrane-embedded carboxylic acid groups in rhodopsin and metarhodopsin II: a Fourier-transform infrared spectroscopy study of site-directed mutants.
    Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10206-10 PMID: 7901852
  32. Mapping light-dependent structural changes in the cytoplasmic loop connecting helices C and D in rhodopsin: a site-directed spin labeling study.
    Biochemistry. 1995 Jul 11;34(27):8812-9 PMID: 7612622
  33. Shift in the relation between flash-induced metarhodopsin I and metarhodpsin II within the first 10% rhodopsin bleaching in bovine disc membranes.
    FEBS Lett. 1981 Dec 28;136(2):201-7 PMID: 7327258
  34. Binding of inositol phosphates to arrestin.
    FEBS Lett. 1991 Dec 16;295(1-3):195-9 PMID: 1765153
  35. Site of attachment of retinal in rhodopsin.
    Nature. 1967 Dec 23;216(5121):1178-81 PMID: 4294735
  36. Complex formation between metarhodopsin II and GTP-binding protein in bovine photoreceptor membranes leads to a shift of the photoproduct equilibrium.
    FEBS Lett. 1982 Jun 21;143(1):29-34 PMID: 6288450
  37. 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.
    Biochemistry. 1996 Nov 12;35(45):14040-6 PMID: 8916888
  38. The carboxyl terminus of bovine rhodopsin is not required for G protein activation.
    Mol Pharmacol. 1994 Dec;46(6):1036-40 PMID: 7808422
  39. Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters.
    Bioconjug Chem. 1993 Mar-Apr;4(2):105-11 PMID: 7873641
  40. Crystal structure of rhodopsin: A G protein-coupled receptor.
    Science. 2000 Aug 4;289(5480):739-45 PMID: 10926528
  41. Direct observation of the thermal equilibria among lumirhodopsin, metarhodopsin I, and metarhodopsin II in chicken rhodopsin.
    Biochemistry. 1994 Nov 29;33(47):14351-8 PMID: 7947845
  42. Photochemical and biochemical properties of chicken blue-sensitive cone visual pigment.
    Biochemistry. 1997 Oct 21;36(42):12773-9 PMID: 9335534
  43. Intrahelical arrangement in the integral membrane protein rhodopsin investigated by site-specific chemical cleavage and mass spectrometry.
    Biochemistry. 2000 Apr 25;39(16):4907-14 PMID: 10769149
  44. Mechanism of rhodopsin kinase activation.
    J Biol Chem. 1991 Jul 15;266(20):12949-55 PMID: 2071581
  45. Temperature and pH dependence of the metarhodopsin I-metarhodopsin II equilibrium and the binding of metarhodopsin II to G protein in rod disk membranes
    Biochemistry. 1999 Jun 29;38(26):8598 PMID: 10387108
  46. Structure and function in rhodopsin: effects of disulfide cross-links in the cytoplasmic face of rhodopsin on transducin activation and phosphorylation by rhodopsin kinase.
    Biochemistry. 1999 Sep 28;38(39):12893-8 PMID: 10504260
  47. Palmitylation of a G-protein coupled receptor. Direct analysis by tandem mass spectrometry.
    J Biol Chem. 1992 Aug 25;267(24):16889-94 PMID: 1512231
  48. Structure and function in rhodopsin: rhodopsin mutants with a neutral amino acid at E134 have a partially activated conformation in the dark state.
    Proc Natl Acad Sci U S A. 1997 Dec 23;94(26):14273-8 PMID: 9405602
  49. Intrinsic biophysical monitors of transducin activation: fluorescence, UV-visible spectroscopy, light scattering, and evanescent field techniques.
    Methods Enzymol. 2000;315:471-89 PMID: 10736721
  50. The structure of mammalian rod opsins.
    Vision Res. 1984;24(11):1501-8 PMID: 6533984
  51. Light-induced binding of 48-kDa protein to photoreceptor membranes is highly enhanced by phosphorylation of rhodopsin.
    FEBS Lett. 1984 Oct 29;176(2):473-8 PMID: 6436059
  52. Light-dependent phosphorylation of rhodopsin: number of phosphorylation sites.
    Biochemistry. 1982 Jun 8;21(12):3014-22 PMID: 6980670
  53. Effect of carboxylic acid side chains on the absorption maximum of visual pigments.
    Science. 1989 Nov 17;246(4932):928-30 PMID: 2573154
  54. Binding of arrestin to cytoplasmic loop mutants of bovine rhodopsin.
    Biochemistry. 1999 Apr 20;38(16):5117-23 PMID: 10213616
  55. Glutamic acid-113 serves as the retinylidene Schiff base counterion in bovine rhodopsin.
    Proc Natl Acad Sci U S A. 1989 Nov;86(21):8309-13 PMID: 2573063
  56. The structure of bovine rhodopsin.
    Biophys Struct Mech. 1983;9(4):235-44 PMID: 6342691
  57. Resonance Raman studies of bovine metarhodopsin I and metarhodopsin II.
    Biochemistry. 1978 Jun 13;17(12):2430-5 PMID: 678522
Article Info
Journal
Biochemistry
Abbr.
Biochemistry
ISSN
0006-2960
DOI
10.1021/bi0018685
Email
imamoto@ms.aist-nara.ac.jp
Published
2000-12-12
Pages
15225-33
Language
English
Region
United States
NLM ID
0370623
PMCID
PMC1382172
Subset
IM
Grants
NEI NIH HHS · R01 EY008061 · United States
NEI NIH HHS · EY08061 · United States
External Links
PubMed source DOI: 10.1021/bi0018685 PMC Full Text View journal details
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