PMC

Pictorial view of the putative binding sites identified using SiteMap in: (A) opsin (B), rhodopsin (11CR), (C) isorhodopsin (9CR), and (D) Meta II.

UV-vis characterization of WT 11CR, G90V 11CR with and without Q treatment and G90V 9CR-Q. Dark state (solid line), photobleaching (dotted line) and acidification (dashed line). Samples in PBS pH 7.4 and 0.05% DM. Spectra were recorded at 20 °C. (A) WT 11CR. (B) G90VT 11CR. (C) G90V 11CR-Q. (D) G90V 9CR-Q.

Absorption spectra of the first elution of immunopurified WT and G90V mutant regenerated with 11-cis-retinal (11CR) and 9-cis-retinal (9CR). After the immunopurification the receptors were characterized by UV-vis spectroscopy. Solid line represents the receptor without treatment, dotted line represents the receptor with 1 µM Q treatment. Samples were eluted with 100 µM of 9-mer peptide in PBS pH 7.4 and 0.05% DM. Spectra were recorded at 20 °C. (A) WT 11CR. (B) WT 9CR. (C) G90V 11CR. (D) G90V 9CR.

Meta II decay of the immunopurified WT and G90V mutant regenerated with 11CR or 9CR with or W/O 1 µM Q treatment. Samples were incubated at 20 °C, and after a steady base line was obtained, they were photobleached and the Trp fluorescence was monitored over time. The fluorescence increase was fit to a single exponential function and the t_1/2 calculated. Mean value and standard error (SE) obtained from independent purifications (n = 3).

Gt activation by WT and G90V mutant regenerated with 11CR or 9CR with or W/O 1 µM Q treatment. Gt activity was measured by means of a radionucleotide filter-binding assay in Gt buffer. The reaction was initiated by the addition of the WT or mutant, and samples were filtrated at different times in the dark and after illumination. (A) WT 11CR with (⚪) and W/O 1 µM Q (●). (B) WT 9CR with (⚪) and W/O 1 µM Q (●). (C) G90V 11CR with (⚪) and W/O 1 µM Q (●). (D) G90V 9CR with (⚪) and W/O 1 µM Q (●). Mean value and standard error (SE) obtained in independent purifications (n = 3).

Chemical stability of the immunopurified WT and G90V mutant. Immunopurified protein samples in PBS pH 7.4 and 0.05% DM, after Q treatment, were incubated with 50 mM hydroxylamine, pH 7 and the decrease of A_max was recorded over time at 20 °C. (A) WT 11CR with (⚪) and W/O 1 µM Q (●). (B) WT 9CR with (⚪) and W/O 1 µM Q (●). (C) G90V 11CR with (⚪) and W/O 1 µM Q (●). (D) G90V 9CR with (⚪) and W/O 1 µM Q (●). Mean value and standard error (SE) obtained in independent purifications (n = 3).

Q is identified in the third elution of immunopurified G90V 9CR mutant sample. During the experiments, a second protein elution was performed to recover as much protein as possible. This second elution was done in PBS pH 6 and used in the regeneration experiments. When it was possible, up to a third elution was carried out in the samples that showed a higher yield, as was the case in the treatments with Q in the receptors regenerated with 9CR. Solid line represents the third elution of G90V 9CR without Q treatment. Dashed line represents the third elution of G90V 9CR-Q with treatment of 1 µM Q. Dotted line represents the difference spectra of G90V 9CR-Q minus G90V 9CR. Samples were eluted with 100 µM of 9-mer peptide in PBS pH 7.4 and 0.05% DM.

Initial rates of chromophore regeneration of the immunopurified WT and G90V mutant with 1 µM Q treatment. (A) 2.5 molar excess fold of 11CR or 9CR (0.925 µM) (dotted line) with regard to Rho concentration (0.37 µM) (solid line) was added to the immunopurified WT and the G90V mutant, in the different buffers containing 0.05% DM + 1 µM Q, samples were illuminated with light of >495 nm to avoid photobleaching of the free retinal (dashed line), and successive spectra were recorded at 20 °C in the dark until no further increase in A_max was detected (inset). (B) The normalized absorbance values at A_max were plotted as a function of time, the data were fit to a single exponential function and the initial rates derived. (C) Initial rates of chromophore regeneration of WT 11CR and WT 9CR. (D) Initial rates of chromophore regeneration of G90V 11CR and G90V 9CR. Mean value and standard error (SE) obtained in independent purifications (n = 3).