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http://dx.doi.org/10.5352/JLS.2012.22.7.863

Rhodopsin Chromophore Formation and Thermal Stabilities in the Opsin Mutant E134Q/M257Y  

Kim, Jong-Myoung (Department of Marine-BioMaterials and Aquaculture, College of Fisheries Sciences, PuKyong National University)
Publication Information
Journal of Life Science / v.22, no.7, 2012 , pp. 863-870 More about this Journal
Abstract
Rhodopsin, a dim light photoreceptor, has been regarded as one of the model systems for the structural and functional study of G protein-coupled receptors (GPCRs). Constitutively active mutant GPCRs leading to the activation of heterotrimeric GDP/GTP-binding protein signaling in the absence of ligand binding are of interest for the study of the activation mechanism in GPCRs. The present study focused on the opsin mutant E134Q/M257Y, which showed a moderate level of constitutive activity and the formation of two distinct rhodopsin chromophores with absorption maxima of 500 nm and 380 nm, depending on the presence of an inverse agonist, 11-cis-retinal, and an agonist, all-trans-retinal, respectively. Reconstitution of the mutant rhodopsin upon incubation with different ratios of 11-cis-retinal and the all-trans-retinal, as well as upon sequential binding of the two retinals, indicated its preferential binding to 11-cis-retinal. The thermal stability of the 11-cis-retinal-bound form of the E134Q/M257Y mutant was lower than that of the mutants containing a single replacement but higher than that of the all-trans-retinal-bound forms. The mutant also showed a lower stability in its opsin state as compared with that of the wild-type opsin but had little effects on the binding affinity to 11-cis-retinal. Information obtained in this study will be helpful for analyzing the structural changes associated with the activation of rhodopsin and GPCRs.
Keywords
G protein-coupled receptor (GPCRs); rhodopsin; opsin; constitutive activation; thermal stability;
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1 Bond, R. A. and Ijzerman, A. P. 2006. Recent developments in constitutive receptor activity and inverse agonism, and their potential for GPCR drug discovery. Trends Pharmacol. Sci. 27, 92-96.   DOI
2 Choe, H. W., Kim, Y. J., Park, J. H., Morizumi, T., Pai, E. F. and Ernst, O. P. 2011. Crystal structure of metarhodopsin II. Nature 471, 651-5.   DOI   ScienceOn
3 Costanzi, S., Siegel, J., Tikhonova, I. G. and Jacobson, K. A. 2009. Rhodopsin and the others: a historical perspective on structural studies of G protein-coupled receptors. Curr. Pharm. Des. 15, 2994-4002.
4 Deupi, X. and Standfuss, J. 2011 Structural insights into agonist-induced activation of G-protein-coupled receptors. Curr. Opin. Struct. Biol. 21, 541-551.   DOI
5 Han, M., Smith, S. O. and Sakmar, T. P. 1998. Constitutive activation of opsin by mutation of methionine 257 on transmembrane helix 6. Constitutive activation of opsin by mutation of methionine 257 on transmembrane helix 6.. Biochemistry 37, 8253-8261.   DOI
6 Hopkins, A. L. and Groom, C. R. 2002. The druggable genome. Nat. Rev. Drug Discov. 1, 727-30.   DOI   ScienceOn
7 Khorana, H. G. 2000. Molecular biology of light transduction by the mammalian photoreceptor rhodopsin. J. Biomol. Struc. Dyn. 11, 1-16.
8 Kim, J. -M., Thurmond, R. L., Altenbach, C., Khorana, H. G. and Hubbell, W. L. 1997. 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. USA 94, 14273-14278.   DOI
9 Kim, J. M., Altenbach, C., Kono, M., Oprian, D. D., Hubbell, W. L. and Khorana, H. G. 2004. Structural origins of constitutive activation in rhodopsin: Role of the K296/E113 salt bridge. Proc. Natl. Acad. Sci. USA 101, 12508-13.   DOI
10 Knowles, A. and Priestley, A. 1978 The preparation of 11-cis-retinal. Vision Res. 18, 115-116.   DOI
11 Molday, R. S. and MacKenzie, D. 1983. Monoclonal antibodies to rhodopsin: characterization, cross-reactivity, and application as structural probes. Biochemistry 22, 653-660.   DOI
12 Oprian, D. D., Molday, R. S., Kaufman, R. J. and Khorana, H. G. 1987. Expression of a synthetic bovine rhodopsin gene in monkey kidney cells. Proc. Natl. Acad. Sci. USA 84, 8874-78.   DOI   ScienceOn
13 Palczewski, K., Kumasaka, T., Hori, T., Behnke, C. A., Motoshima, H., Fox, B. A., LeTrong, I., Teller, D. C., Okada, T., Stenkamp, R. E., et al. 2000 Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289, 739-745.   DOI   ScienceOn
14 Papermaster, D. S. 1982. Preparation of antibodies to rhodopsin and the large protein of rod outer segment. Methods Enzymol. 81, 240-246.   DOI
15 Park, J. H., Scheerer, P., Hoffmann, K. P., Choe, H. W. and Ernst, O. P. 2008. Crystal structure of the ligand-free G-protein-coupled receptor opsin. Nature 454, 183-187.   DOI   ScienceOn
16 Parnot, C., Miserey-Lenkei, S., Bardin, S., Corvol, P. and Clauser, E. 2002. Lessons from constitutively active mutants of G protein-coupled receptors. Trends Endocrinol. Metab. 13, 336-343.   DOI
17 Rao, V. R. and Oprian, D. D. 1996. Activating mutations of rhodopsin and other G protein-coupled receptors. Annu. Rev. Biophys. Biomol. Struct. 25, 287-314.   DOI
18 Sakmar, T. P., Franke, R. R. and Khorana, H. G. 1989. Glutamic acid-113 serves as the retinylidene Schiff base counterion in bovine rhodopsin. Proc. Natl. Acad. Sci. USA 86, 8309-8313.   DOI   ScienceOn
19 Rasmussen, S. G., DeVree, B. T., Zou, Y., Chung, K. Y., Kobilka, T. S., et al. 2012. Crystal structure of the beta(2) adrenergic receptor-Gs protein complex. Nature 477, 549-555.
20 Reeves, P. J., Hwa, J. and Khorana, H. G. 1999. Structure and function in rhodopsin: Kinetic studies of retinal binding to purified opsin mutants in defined phospholipid-detergent mixtures serves as probes of the retinal binding pocket. Proc. Natl. Acad. Sci. USA 96, 1927-31.   DOI
21 Sambrook, J. and Russell, D. W. 2001. Molecular cloning: A laboratory manual. 3rd edition. Cold Spring Harbor Laboratory Press, NY, Plainview.
22 Standfuss J., Edwards, P. C., D'Antona, A. and Schertler, G. F. X. 2011. The structural basis of agonist-induced activation in constitutively active rhodopsin. Nature 471, 656-661.   DOI
23 Xie, G., Gross, A. K. and Oprian, D. D. 2003. An opsin mutant with increased thermal stability. Biochemistry 42, 1995-2001.   DOI
24 Xu, F., Wu, H., Katritch, V., Han, G. W., Jacobson, K. A., Gao, Z. G., Cherezov, V. and Stevens, R. C. 2011. Structure of an agonist-bound human A2A adenosine receptor. Science 332, 322-327.   DOI