The Hydrodynamic Properties of Dark- and Light-activated States of n-Dodecyl β-D-Maltoside-solubilized Bovine Rhodopsin Support the Dimeric Structure of Both Conformations

Abstract

Rhodopsin (Rho) has been extracted in n-dodecyl beta-D-maltoside (DM) from bovine retinal rod outer segments and purified to homogeneity by affinity chromatography on concanavalin A-Sepharose. Because chemical cross-linking of Rho and photoactivated Rho (Rho*) provided initial evidence for the oligomeric nature of the photoreceptor protein, we carried out a hydrodynamic characterization of the native and activated conformations of detergent-solubilized Rho. The molecular weights of the complexes between dark and photoexcited states of Rho and DM were determined by gel filtration chromatography on Sephacryl S-300, in the presence of 0.1% DM. Subtracting the size of the corresponding detergent micelles resulted in molecular masses of 78 kDa for native Rho and 76 kDa for Rho*. The measured content of 0.97 g of detergent/g of protein resulted in a calculated partial specific volume of 0.765 cm(3)/g for the protein-detergent complex and a molar mass of 64-65 kDa for the protein moiety. The sizes of Rho.DM and Rho*.DM complexes were also evaluated by sedimentation on 10-30% sucrose gradients, in the presence of 0.1% DM, and molecular masses of about 60 kDa were estimated for both the dark- and light-activated states of the photoreceptor protein. The size of Rho was determined to be 65,300 and 69,800 Da, respectively, when the purified Rho.DM complex was either chromatographed on Sephacryl S-300 or ultracentrifuged on sucrose gradients in the absence of DM. All these results were consistent with a dimeric quaternary structure for both conformations of Rho. Additionally, the functional integrity of the purified photoreceptor protein following gel filtration chromatography and ultracentrifugation was demonstrated by three criteria as follows: (i) its characteristic UV-visible absorption spectra, (ii) its capability to photoactivate transducin, and (iii) its ability to serve as a substrate for rhodopsin kinase.