We used steady-state and time-resolved (pulsed) fluorescence spectroscopies to study the dynamic structure of the COOH terminus in bacteriorhodopsin. The extrinsic fluorophore 8-aminonaphthalene-1,3,6-trisulfonic acid was attached to the protein, in purple membrane sheets, with a water-soluble carbodiimide; about 50% of the dye molecules were found to be attached to the COOH terminus. Signals from samples treated with papain were subtracted from non-papain-treated sample signals to give information about the COOH-terminus dynamics. Dye molecules on the COOH terminus fluoresce more strongly than dye molecules bound elsewhere on the membrane. This result, combined with our calculations showing that retinal is an energy acceptor in our system, suggests that the C-terminus spends most of its time away from the membrane surface. We systematically studied the effects of temperature, ionic strength, and pH, fitting the time-resolved anisotropy to r(t) = r∞ + (r0 - r∞)e-t/φ; the following picture emerged: In the pH range 5.6-10.9, raising the pH has the effect of increasing the fluorescence intensity and decreasing r∞. We interpret this result to mean that the time-averaged position of the COOH terminus becomes farther from retinal as the pH is raised and that the range of Brownian motions of the C-terminus increases along with the pH. At pH 6.6, adding NaCl up to a concentration of 10 mM had qualitatively the same effects as raising the pH. Over the temperature range 10-50°C, the time constant for anisotropy decay scales closely with the viscosity of water, a result consistent with Brownian motions of the COOH terminus in bulk water. We invariably found that r0 > 2r∞. All of our results are consistent with the conclusion of Wallace and Henderson [Wallace, B. A., & Henderson, R. (1982) Biophys. J. 39, 233] that the COOH terminus of bacteriorhodopsin is free to assume many positions.
|Number of pages||5|
|Publication status||Published - 1986|
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