Spectral comparisons
Béjà et al. reported an absorbance maximum for pR of 520 nm in E. coli membranes, using a difference bleaching technique to remove interfering absorbance bands from other membrane components in the impure pR sample [1]. We confirmed this result using crude E. coli membranes without detergent present (data not shown). However, we observed an absorbance maximum of 546 ± 5 nm for pR in OG at pH 7 at all stages of purification (Fig. 1). Small blue shifts were observed for pR samples in OG when measured at pH 8 and 9.5 (8 and 16 nm, respectively; data not shown).
For the pR samples reconstituted in DHPC, which were used for flash photolysis experiments, the absorbance maxima were similar to those measured in OG (spectra not shown). A chromophore absorption maximum near 540 nm was also obtained by using difference spectroscopy of pR in crude E. coli membranes solubilized in OG (spectrum not shown). However, for pR measured directly in crude E. coli membranes, i.e. not solubilized in OG, we obtained the same value (520 nm) as reported previously [1].
Solubilization in detergent presumably leads to structural distortions of the native protein conformation, and therefore a change in the absorbance properties of the chromophore. However, the direction that λmax for pR would have to shift upon solubilization in OG is inconsistent with the pattern for bR, whose λmax decreases when it is solubilized in OG [12]. Furthermore, pR in OG showed resonance Raman spectra (D. Dunmire, R. A. Krebs and M. S. Braiman, unpublished data) indicative of a chromophore structure very close to native light-adapted (i.e. all-trans) bR in purple membrane. However, there is one major difference: pR in OG exhibits an upshifted, doublet C=N Schiff base band consisting of two components of nearly-equal intensity. These appear to correspond to the presence of two distinct subpopulations of pR, at least when expressed in E. coli and solubilized in OG micelles. The different values of λmax for pR in membrane state [1] and OG solution might be related to the presence of these multiple subpopulations, but this connection remains unclear.