Figure 2.

Photointermediate kinetics and proton release in the E134R/R135E mutant of rhodopsin. The upper left panel shows the time-resolved absorbance changes observed after photolysis. Data was collected at (heavy lines) 30, 60, 120, 240, 400, 1000ns (fine lines), 10, 30, 50, 100, 150, 200, 300, 400, 800 μs, 2 and 10 ms. The increase in absorbance at 30 ns, peaking near 560 nm, is due to formation of the Batho intermediate. The increase in absorbance at 380 nm developing at the longer times is due to the final photointermediate on this time scale, Meta II. The negative absorbance change at long times near 500 nm is due to the disappearance of the pigment absorbance (and other protonated Schiff base intermediates) after photolysis. The spectral changes seen in this mutant are essentially identical to those seen after photolysis of a wild type rhodopsin sample solubilized in detergents such as dodecyl maltoside (15,20). The b-spectra obtained from the E135R/R135E mutant are presented in the upper right panel and show the spectral changes associated with each exponential time constant as determined from the global exponential fitting process (see text), and the b-spectrum labeled 0 is the time independent spectral change seen here. Points in the figure give the experimental b-spectra, and the smooth curves show the fit to the data using microscopic rate constants and model intermediate spectra determined from fitting. The observed lifetimes associated with the time dependent components (1, 2, 4 and 5) are given in Table 1. The quality of the fit to these four exponentials is shown by the residuals (difference between the actual data and fit computed using the time constants and b-spectra) plotted in the lower right panel (traces have been offset by 0.005 AU, shortest time delay is at bottom). The lower left panel shows time resolved absorbance changes seen at late times in the presence of BCP. Delay times shown are 50, 100, 200, 500 μs, 1, 2, and 10 ms. At these times, long after Batho has disappeared, the changes in the red, near 595 nm, are due to disappearance of the deprotonated form of BCP at late times (i.e. proton release by rhodopsin). The protonation changes in E134R/R135E are the opposite of what is observed under these conditions in wild type rhodopsin. For comparison of kinetics with other mutants, the 2 ms time-resolved absorbance spectra are shown as dashed lines.