ABC  Vol.2 No.2 , May 2012
Digitonin and sodium dodecylsulfate-solubilized frog rho-dopsin: Behavior under native and denaturing polyacrylamide gel electrophoresis
ABSTRACT
Rhodopsin oligomerization and dissociation in vivo and under experimental conditions is an important topic both for a basic understanding of photoreceptor structure-function but also as a potential eye disease mechanism. In this study, to estimate a state rhodopsin after solubilization with mild and harsh detergents, we applied the native (blue native-PAGE, BN- PAGE) and denaturing electrophoresis (blue-urea- PAGE, BU-PAGE; blue-SDS-PAGE, BSDS-PAGE and SDS- PAGE). After blue BN-PAGE and BSDS- PAGE, rhodopsin and opsin, respectively, were presented in gels a major band of dimer with slight contents of higher oligomers without any traces of monomer, thus testifying in favor dimer-heteromeric state of frog rhodopsin in the photoreceptor membrane. Despite all oligomer bands gave positive staining with the rhodopsin-specific monoclonal antibodies (mAb), subsequent SDS-PAGE in combination with electroelution in denaturing conditions showed that stained bands are not homogenous and besides of opsin oli-gomers contain a small admixture of proteins with unknown function. Unfolding of opsin oligomers by solubilization in SDS, as compared with folded opsin in digitonin, induces their transition to a more compact conformation. It was manifested in a more rapid migration of opsin oligomers toward to anode. Cooling of digitonin/SDS mixed extracts at 4?C for 24 hours led to a partial reverse transition of unfolded opsin dimer to initial folded conformation, thus de- monstrating the entropic nature of this transition. Opsin monomer can be observed in the gels only after harsh dissociation of oligomers under BU-PAGE or SDS-PAGE. The electro elution of the individual opsin oligomers with denaturing buffer followed by SDS-PAGE resulted in dissociation of dimer to monomers. However, unexpectedly, the trimer was dissociated to a prevailing dimer and a small portion of monomer. The products dissociation of both opsin tetramer and pentamer are difficult to determine precisely, but they are neither monomer nor dimer. Dissociation data show that the degree of opsin oli- gomerization by unknown reasons affects the pattern of dissociation of its aggregates. Obtained in this paper data indicate a need for further detailed study the obscure mechanisms of aggregation-dissociation of rhodopsin.

Cite this paper
Shukolyukov, S. (2012) Digitonin and sodium dodecylsulfate-solubilized frog rho-dopsin: Behavior under native and denaturing polyacrylamide gel electrophoresis. Advances in Biological Chemistry, 2, 84-91. doi: 10.4236/abc.2012.22011.
References
[1]   Blasie, J.K. and Worthington, C.R. (1969) Molecular localization of frog retinal receptor photopigment by electron microscopy and low-angle X-ray diffraction. Journal of Molecular Biology, 39, 407-416. doi:10.1016/0022-2836(69)90135-1

[2]   Chabre, M. (1975) X-ray diffraction studies of retinal rods. I. Structure of the disc membrane, effect of illumination. Biochimica et Biophysica Acta, 382, 322-335. doi:10.1016/0005-2736(75)90274-6

[3]   Edrington, T.C., Bennett, M. and Albert, A.D. (2008) Calorimetric studies of bovine rod outer segment disk membranes supports a monomeric unit for both rhodopsin and opsin. Biophysical Journal, 95, 2859-2866. doi:10.1529/biophysj.108.128868

[4]   Chabre, M., Deterre, P. and Antonny B. (2009) The apparent cooperativity of some GPCRs does not necessarily imply dimerization. Trends in Pharmacological Sciences, 30, 188-197. doi:10.1016/j.tips.2009.01.003

[5]   Fotiadis, D., Liang Y. and Filipek S., et al. (2003) Atomic force microscopy: Rhodopsin dimers in native disc membranes. Nature, 421, 127-128. doi:10.1038/421127a

[6]   Fotiadis, D., Liang, Y. and Filipek S., et al. (2003) The G protein-coupled receptor rhodopsin in the native membrane. FEBS Letters, 564, 281-288. doi:10.1016/S0014-5793(04)00194-2

[7]   Liang, Y., Fotiadis D. and Liang Y., et al. (2003) Organization of the G protein-coupled receptors rhodopsin and opsin in native membranes. The Journal of Biological Chemistry, 278, 21655-21662. doi:10.1074/jbc.M302536200

[8]   Filipek, S., Krzysko, K.A. and Fotiadis, D., et al. (2004) A concept for G protein activation by G protein-coupled receptor dimers: The transducin/rhodopsin interface. Photochemical & Photobiological Sciences, 3, 628-638. doi:10.1039/b315661c

[9]   Jastrzebska, B., Fotiadis, D. and Jang, G.F., et al. (2006) Functional and structural characterization of rhodopsin oligomers. The Journal of Biological Chemistry, 281, 11917-11922. doi:10.1074/jbc.M600422200

[10]   Morris, M.B., Dastmalchi, S. and Church, W.B. (2009) Rhodopsin: Structure, signal transduction and oligomerisation. The International Journal of Biochemistry & Cell Biology, 41, 721-724. doi: 10.1016/j.biocel.2008.04.025

[11]   Gurevich, V.V. and Gurevich, E.V. (2008) How and why do GPCRs dimerize? Trends in Pharmacological Sciences, 29, 234-240. doi:10.1016/j.tips.2008.02.004

[12]   Milligan, G. (2009) G protein-coupled receptor hetero-dimerization: Contribution to pharmacology and function. British Journal of Pharmacology, 158, 5-14. doi:10.1111/j.1476-5381.2009.00169.x

[13]   Chamber, M., Deterred, P. and Antonny, B. (2009) The apparent cooperativity of some GPCRs does not necessarily imply dimerization. Trends in Pharmacological Sciences, 30, 188-197.

[14]   Liu, Z., Zhang, J. and Zhang, A. (2009) Design of multivalent ligand targeting G-protein-coupled receptors. Current Pharmaceutical Design, 15, 682-718. doi:10.2174/138161209787315639

[15]   Medina, R., Promo, D. and Bubis, J. (2004) The hydrodynamic properties of dark- and light-activated states of n-dodecyl-beta-D-maltoside solubilized bovine rhodopsin support the dimeric structure of both confomations. The Journal of Biological Chemistry, 279, 39565-39573. doi:10.1074/jbc.M402446200

[16]   Shukolyukov, S.A. (2009) Aggregation of frog rhodopsin to oligomers and their dissociation to monomer: Application of BN- and SDS-PAGE. Biochemistry (Moscow), 74, 599-604. doi:10.1134/S0006297909060029

[17]   Shukolyukov, S.A. (2010) Proof of oligomeric state of frog rhodopsin: Visualization of dimer and oligomers on gels after BN- and HRCN-PAGE using Antibodies to rhodopsin and by retinylopsin fluorescence. Biochemistry (Moscow), 75, 1045-1051. doi:10.1134/S0006297910080146

[18]   Neri, M., Vanni, S., Tavernelli, I. and Rothlisberger, U. (2010) Role of aggregation in rhodopsin signal transduction. Biochemistry, 49, 4827-4832. doi:10.1021/bi100478j

[19]   Wittig, I., Braun, H.P. and Sch?gger, H. (2006) Blue native PAGE. Nature Protocols, 1, 418-428. doi:10.1038/nprot.2006.62

[20]   Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685. doi:10.1038/227680a0

[21]   Sch?gger, H. (2006) Tricine-SDS-PAGE. Nature Protocols, 1, 16-22. doi:10.1038/nprot.2006.4

[22]   Lowry, O.H., Rosenbrough, N.I., Farr, A.L. and Randall, R.J. (1951). Protein measurement with Folin phenol reagent. The Journal of Biological Chemistry, 193, 265-290.

[23]   Jastrzebska, B., Maeda, T. and Zhu, L., et al. (2004) Functional characterization of rhodopsin monomers and dimers in detergents. The Journal of Biological Chemistry, 279, 54663-54675. doi:10.1074/jbc.M408691200

[24]   Dutta, A., Tirupula, K.C. and Alexiev, U., et al. (2010). Characterization of membrane protein non-native states. 1. Extent of unfolding and aggregation of rhodopsin in the presence of chemical denaturants. Biochemistry, 49, 6317- 6328. doi:10.1021/bi100338e

[25]   Dutta, A., Kim, T.Y., Moeller, M. and Wu, J., et al. (2010) Characterization of membrane protein non-native states. 2. The SDS-unfolded states of rhodopsin. Biochemistry, 49, 6329-6340. doi:10.1021/bi100339x

[26]   Niepmann, M. and Zheng, J. (2006) Discontinuous native protein gel electrophoresis. Electrophoresis, 27, 3949-3951. doi:10.1002/elps.200600172

 
 
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