Abstract

MEDINA, Rafael; MOLLER, Carolina; PERDOMO, Deisy  and  BUBIS, José. Aproximaciones de bioquímica clásica al estudio de la relación entre la estructura y la función de la rodopsina . AVFT [online]. 2008, vol.27, n.1, pp.5-13. ISSN 0798-0264.

The photoreceptor protein rhodopsin (R) was extracted from bovine retinal rod outer segments using the detergent n-dodecyl β-D-maltoside (DM), and purified to homogeneity by affinity chromatography. Chemical cross-linking of R and photoactivated rhodopsin (R*) with the bifunctional agents sulfo-succinimidyl 4-(N-maleimidomethyl) ciclohexane-1-carboxylate (sulfo-SMCC) or m-maleimidobenzoyl-N-hydroxysuccinimide ester, suggested the oligomeric nature of this photoreceptor protein. The characterization of the hydrodynamic parameters of R and R* in the presence of 0.1% DM, using molecular exclusion chromatography and sedimentation on sucrose gradients, allowed us to estimate the size of the R:DM and R*:DM complexes. Our results agreed with a dimeric quaternary structure for both, R and R*. R crosslinked with sulfo-SMCC, in the presence of light, was stabilized in a photointermediate that absorbed at ~ 470 nm. Experiments of proteolysis with termolysine on R native dimers and R monomers generated by using high concentrations of DM, together with modeling studies based on the reported crystal structure of the protein, suggested that sulfo-SMCC generated an intramolecular cross-link between Cys₁₄₀ and Lys₂₄₈ of R, which was probably responsible for the incapacity of the protein to display the conformational change required to reach its photoactivated state.

Keywords : Rhodopsin; receptor dimerization; chemical cross-linking; photointermediates; visual process.