Fourier transform infrared study of the N intermediate of bacteriorhodopsin

Abstract

Visible absorption spectroscopic experiments show that the N intermediate is the main photoproduct of a highly hydrated film of the light-adapted bacteriorhodopsin (70% water by weight) at pH 10 and 274 K. The difference Fourier transform infrared spectrum between the N intermediate and unphotolyzed light-adapted bacteriorhodopsin was recorded under these conditions. A small amount of the M intermediate present did not affect this spectrum significantly. The difference spectrum exhibited a positive band at 1755 cm-1 (probably due to Asp-85) and a negative band at 1742 cm-1 (due to Asp-96), neither of which was observed for the M intermediate. The spectrum of the N intermediate at pH 7 was nearly identical with that at pH 10. Spectra at pH 10 also were measured with isotope-substituted samples. A vibrational band at 1692 cm-1 due to the peptide bond disappeared, and a band at 1558 cm-1 emerged upon formation of the N intermediate. The spectrum also displayed bands containing the N-H and C15-H in-plane bending vibrational modes at 1394 and 1303 cm-1. These frequencies are similar to those of the L intermediate while the intensities of these bands are larger than those in the L intermediate, suggesting that the Schiff bases of both the L and N intermediates have a strong hydrogen-bonding interaction with the protein and that the C12-H to C15-H region of the chromophore is less twisted in the N intermediate than in the L intermediate.

Trapping the M sub 1 and M sub 2 substrates of bacteriorhodopsin for electron diffraction studies

“…with the report that close to 100% M is trapped at 230 K for humidified PM samples (Pfefferle et al, 1991). Because the cold nitrogen gas can dry the specimen over the course of hours, I found it necessary to rehydrate the specimen after each session of measurements.…”

“…The isosbestic point for bR and M is at 455 nm, and is more clearly defined than the isosbestic point occuring at the same wavelength in the spectra taken at 260 K. By focusing more light on the sample, I went from consistently obtaining~80 % M to greater than 90 % M at 260 K. lt is possible that a greater % of M could be "trapped" at 260 K with a stronger actinic light source. However, Ormos (1991) reports that there is a significant N-intermediate component in his M -bR difference spectra taken at 260 K. As judged by the characterization of the N intermediate (Pfefferle et al, 1991), it may be that there is a greater concentration of N than M in the Ormos 260 K difference spectra (See the discussion in Chapter 6.). This can be explained by 4 -the models that best fit the bR photocycle kinetics, which allow for the thermal decay of M2 to N and the back-reaction of N to M2 _ Varo & Lanyi, 1990).…”

“…This is a reversible first-order reaction (Bray & White, 1966). The rate equations are, .100 95 Roepe et al, 1987;Gerwert et al, 1989;Bousche et al, 1991;Braiman et al, 1991;Pfefferle et al, 1991). (Bell, 1972).…”

“…cm "1, 1186 cm "1 and 1396 cm "1 (Pfefferle et al, 1991, close to the L-bands, it is best not to use these three B bands as L-marker bands.…”

“…In my 240 K spectra, the absence of a " negative 1739 cm "1 peak is conspicuous; if there is a positive 1738 cm "1 peak, it is buried in the noise. Moreover, the presence of a strong, positive 1762 cm"1 band, which is used to characterize M (Pfefferle et al, 1991) speaks strongly that M is trapped with little if any L present.…”