Transmembrane ionic transport is a fundamental aspect of many physiological processes. To understand transport phenomena at a molecular level, structural information on the dynamic, conformational changes of active transport proteins is required. Ideally, one would determine protein structural changes associated with individual, elementary steps in the ion transport reaction. Few techniques exist for obtaining such information. A fluorescence energy transfer technique has been developed in our laboratory that provides specific structural information on membrane-protein dynamics. Phase Modulation of Fluorescence Energy Transfer has been used in initial studies to quantitatively measure changes in the location of the retinal in bacteriorhodopsin during its proton-pumping photocycle. Fluorescence energy transfer can be measured from lipid donors to photocycle intermediates. The population of the photocycle intermediate is modulated by mechanically chopping the actinic light driving bacteriorhodopsin's photosystem. The resulting quenching of fluorescence due to energy transfer from the lipid donor to a specific photocycle intermediate acting as an acceptor is measured with phase-sensitive detection. The donor fluorescence and acceptor absorbance amplitudes can be analyzed to determine the distance of closest approach of the lipid donor to the absorbing acceptor species in the photocycle intermediate. Thus, highly specific information concerning the structure of reaction intermediates in the proton pumping process is obtained. The proposed study is to extend our previous work to a more detailed study of bacteriorhodopsin (bR) and to new studies on halorhodopsin (hR) and sensory rhodopsin(sR). In the bR work, a variety of fluorescent donors will be used to locate the position of retinal in the two M intermediate states. Preliminary studies show a significant difference between the retinal location in the two M states. Fluorescent donors located at different depths in the lipid bilayer will be investigated. Parallel studies will be initiated on hR and sR. This provides a useful comparison to bR and will establish the generality of the bR results. Because the absorbance maxima of the photocycle intermediates of hR and sR are well separated, these proteins offer a better system for the application of this energy transfer technique. Experiments on these proteins should allow a visualization of structural changes at two consecutive steps in the photocycle. Thus, this technique should provide unique structural information on the dynamics of membrane-bound proteins.
| Dewey, T G; Datta, M M (1989) Determination of the fractal dimension of membrane protein aggregates using fluorescence energy transfer. Biophys J 56:415-20 |
| Hasselbacher, C A; Spudich, J L; Dewey, T G (1988) Circular dichroism of halorhodopsin: comparison with bacteriorhodopsin and sensory rhodopsin I. Biochemistry 27:2540-6 |
