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. Author manuscript; available in PMC: 2008 Aug 29.
Published in final edited form as: Biochemistry. 2006 May 2;45(17):5430–5439. doi: 10.1021/bi0525775

Figure 4.

Figure 4

Photointermediate kinetics and proton release/uptake in the E122Q and H211F mutants of rhodopsin. The upper left panel shows the time-resolved absorbance changes observed after photolysis of E122Q. Data were collected at (heavy lines) 30, 90ns, (fine lines)1, 10, 50, 150, 300, 800 μs, 2, 10, 60 and 690 ms. The kinetics of Schiff base deprotonation seen after photolysis of E122Q showed significant slowing relative to wild type rhodopsin under these conditions. This is demonstrated by the time constants given in Table 1 associated with the b-spectra shown in the lower left panel (b-spectrum associated with Batho decay not shown). Time-resolved difference spectra that were collected from E122Q in the presence of BCP at delay times (fine lines) 50, 150, 450 μs, 1, 2, 5, 10, 60 and (heavy line) 690 ms are shown in the left, middle panel. For E122Q, early proton release (bleaching near 595 nm at 450 μs) followed by net proton uptake (absorbance peaking near 595 nm at 690 ms) was seen which is qualitatively similar behavior to wild type rhodopsin. The upper right panel shows the time-resolved absorbance changes observed after photolysis of H211F. Data was collected at (heavy lines) 30, 90ns, (fine lines)1, 10, 50, 300 μs, 2, 10, 30, 60 and 690 ms. The kinetics of Schiff base deprotonation seen after photolysis of H211F showed much greater slowing relative to wild type rhodopsin under these conditions than do those of E122Q. As is shown by the b-spectra in the lower right panel, for H211F the slowest component (6) which was small for E122Q and does not occur in detergent solubilized wild type rhodopsin, is the largest amplitude b-spectrum (b-spectrum associated with Batho decay not shown). In the middle, right panel time-resolved difference spectra are shown that were collected from H211F in the presence of BCP at delay times 300 μs, 2, 8, 25, 55 and 690 ms. For H211F, similarly to E135R/R135E and E134Q (data not shown) only proton release is seen. For comparison of kinetics, the 2 ms time-resolved absorbance spectra are shown as dashed lines.