Orientation of the bacteriorhodopsin chromophore probed by polarized Fourier transform infrared difference spectroscopy

Abstract

Polarized, low-temperature Fourier transform infrared (FTIR) difference spectroscopy has been used to investigate the structure of bacteriorhodopsin (bR) as it undergoes phototransitions from the light-adapted state, bR570, to the K630 and M412 intermediates. The orientations of specific retinal chromophore and protein groups relative to the membrane plane were calculated from the linear dichroism of the infrared bands, which correspond to the vibrational modes of those groups. The linear dichroism of the chromophore C=C and C-C stretching modes indicates that the long axis of the polyene chain is oriented at 20-25 degrees from the membrane plane at 250 K and that it orients more in-plane when the temperature is reduced to 81 K. The polyene plane is found to be approximately perpendicular to the membrane plane from the linear dichroism calculations of the HOOP (hydrogen out-of-plane) wags. The orientation of the transition dipole moments of chromophore vibrations in the K630 and M412 intermediates has been probed, and the dipole moment direction of the C=O bond of an aspartic acid that is protonated in the bR570----M412 transition has been measured.

Conserved amino acids in F‐helix of bacteriorhodopsin form part of a retinal binding pocket

“…The polyene chain was tilted slightly towards the extracellular side of the membrane and the polyene plane was arranged approximately perpendicular to the membrane plane. This orientation is in agreement with polarized visible [16,17], FTIR [18,19], resonance Raman [20] and neutron diffraction [21] measurements. It was assumed that the C9-and C12-methyl groups of retinal were pointed towards the cytoplasmic side of the membrane and the NH bond towards the extracellular medium in agreement with recent evidence (R. Mathies, personal communication; M. Heyn, personal communication).…”

Conformational Changes Detected in a Sensory Rhodopsin II-Transducer Complex

“…1, inset) most likely corresponds to the CϭN stretch of the protonated Schiff base, which downshifts as expected to near 1633 cm Ϫ1 in D 2 O (32). The negative band at 1664 cm Ϫ1 is of particular interest because earlier studies on BR using polarized FTIR associate a negative band at a slightly higher frequency (1669 cm Ϫ1 ) to a small outward tilting (away from the membrane normal) of the ␣-helical structure (40).…”

“…Further progress should be possible by combining FTIR with methods of band assignment including isotope labeling and site-directed mutagenesis. In addition, it should be possible to apply specialized FTIR techniques including polarization (40,58) and attenuated total reflection (52) to probe changes in orientation of specific groups under well defined aqueous environment.…”

Tight Asp-85–Thr-89 association during the pump switch of bacteriorhodopsin

“…Polarized FTIR spectroscopy can characterize hydrogen bonds in terms of both vibrational frequencies and dipole orientations (22)(23)(24). In BR specifically, our highly accurate measurement of K minus BR difference spectra throughout the whole mid-IR (4,000-700 cm…”