Retinal isomer ratio in dark-adapted purple membrane and bacteriorhodopsin monomers

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Abstract
On the basis of data obtained by spectroscopic analysis and chromatography of retinal extracts, a consensus has been adopted that dark-adapted purple membrane (pm) contains 13-cis- and all-trans-retinal in equal amounts, whereas the light-adapted membrane contains all-trans-retinal only. We have developed an improved extraction technique which extracts up to 70% of the retinal in pm within 4 min. In the extracts from dark-adapted pm at room temperature, we consistently find 66-67% 13-cis-retinal and 33-34% all-trans-retinal, and more than 98.5% all-trans isomer in light-adapted samples. The spectrum obtained by reconstitution of bacterioopsin with 13-cis-retinal at 2.degree. C (to minimize isomerization) shows an absorbance maximum at 554 nm and agrees well with the spectrum for the 13-cis component calculated from the dark-adapted and light-adapted bR spectra with our extraction data. The ratio of 13-cis-:all-trans isomer in dark-adapted pm is 2:1 and nearly constant between 0 and 38.degree. C but begins to decrease distinctly above 40.degree. C, and more rapidly near 70.degree. C, reaching 0.75 at 90.degree. C. The van''t Hoff plot of the isomer ratio shows a nonlinear temperature dependence above 40.degree. C, indicating a more complex system than a simple thermal 13-cis/all-trans isomer equilibrium. We attribute the broadening, absorbance decrease, and blue shift of the visible absorption band with increasing temperature to the appearance of at least one and possibly two or three new chromophores which contain, mainly or exclusively, the all-trans isomer. The 2:1 ratio of the isomers and its small temperature dependence between 0 and 40.degree. C suggest that it could be determined by the trimeric association of bR in the planar protein lattice of pm and that the lowest free energy conformation in the lattice is a timer which contains one all-trans and two 13-cis isomers. However, the lattice remains intact up to .apprx. 75.degree. C, and trimers of bR exist at still higher temperatures. Moreover, monomeric bR solubilized with Triton X-100 shows a similar isomer ratio near room temperature and slightly higher 13-cis isomer content (e.g., 71% 13-cis and 29% all-trans at 4.degree. C) at low temperature, and also a nonlinear but steeper temperature dependence in the van''t Hoff plot. We argue that the lattice stabilizes the protein in a conformation which slightly favors the 13-cis chromophore but restricts its structure from assuming the conformation still more favorable to 13-cis-retinal which it attains in the monomer.