Circular dichroism of halorhodopsin: comparison with bacteriorhodopsin and sensory rhodopsin I

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Abstract
Circular dichroic (CD) spectra of three related pigments from Halobacterium halobium, halorhodopsin (HR), bacteriorhodopsin (BR), and sensory rhodopsin I (SR-I), are compared. In native membranes the two light-driven ion pumps, HR and BR, exhibit bilobe circular dichroism spectra characteristics of exciton splitting in the region of retinal absorption, while the phototaxis receptor, SR-I, exhibits a single positive band centered at the SR-I asborbance maximum. This indicates specific aggregation of protein monomers of HR, as previously noted [Sugiyama, Y., and MUkohata, Y. (1984) J. Biochem. (Tokoyo) 96, 413-420], a similar to the well-characterized retinal/retinal exciton interaction in the purple membrane. The absence of this interaction in SR-I indicates SR-I is present in the native membrane as monomers or that interactions between the retinal chromophores are weak due to chromophore orientation or separation. Solubilization of HR and BR with nondenaturing detergents eliminates the exciton coupling, and the resulting CD spectra share simlar features in all spectral regions from 250 to 700 nm. Schiff-base deprotonation of both BR and HR yields positive CD bands near 410 nm and shows similar fine structure in both pigments. Removal of detergent restores the HR native spectrum. HR differs from BR in that circular dichroic bands corresponding to both amino acid and retinal environments are much more sensitive to external salt concentration and pH. A theoretical analysis of the excitron spectra of HR and BR that provides a range of interchromophore distances and orientations is performed. By use of a trimer model for BR, chromophore-chromophore separations are found to be in close agreement with neutron and electron diffraction data. The HR data could be fit to either a dimer or trimer model. The dimer fit gives interchromophore distances closer to that of BR than the trimer fit.