We have used fluorescence energy transfer in the rapid-diffusion limit (RDL) to estimate the trans-membrane depth of retinal in the purple membrane (PM). Chelates of Tb(III) are excellent energy donors for the retinal chromophore of PM, having a maximum Ro value for Förster energy transfer of approximately 62 Å (assuming a donor quantum yield of 1). Energy transfer rates were measured from the time-resolved emission kinetics of the donor. The distance of closest approach between chelates and the chromophore was estimated by simulating RDL energy-transfer rate constants according to geometric models of either PM sheets or membrane vesicles. The apparent rate constant for RDL energy transfer between Tb(III)HED3A and retinal in PM sheets is 1·5(±0·1) × 106 m-1 s-1, corresponding to a depth of approximately 10±2 Å for the retinal chromophore. Cell envelope vesicles (CEVs) from Halobacterium halobium were studied by using RDL energy transfer to assess the proximity of retinal to either the extracellular or intracellular face of the PM. The estimated depth of retinal from the extravesicular face of the PM is 10 ± 3 Å, based on the RDL energy-transfer rate constant. Energy-transfer levels to retinal in the PM were estimated by an indirect method with energy donors trapped in the innerr-aqueous space of CEVs. The rate constants derived for this arrangement are too low to be consistent with the shortest depth of retinal deduced for PM sheets. Thus, the intravescicular face of CEVs, corresponding to the cytoplasmic face of cells, is the more distant surface from the chromophore of bacteriorhodopsin.
Bibliographical noteFunding Information:
This work was supported by grants from the National Institutes of Health (GM27906, AM32961 and RR01439). D.D.T. was supported by an Established Investigatorship from the American Heart Association. R.O.L. was a doctoral dissertation fellow of the University of Minnesota Graduate school.