High-pressure near-infrared Raman spectroscopy of bacteriorhodopsin light to dark adaptation

Abstract

Near-infrared (NIR) Raman spectroscopy is employed as an in situ probe of the chromophore conformation to study the light to dark-adaptation process in bacteriorhodopsin (bR) at variable pressure and temperature in the absence of undesired photoreactions. In dark-adapted bR deconvolution of the ethylenic mode into bands assigned to the all-trans (1526 cm-1) and 13-cis (1534 cm-1) isomers yields a 13-cis to all-trans ratio equal to 1 at ambient pressure (Schulte et al., 1995, Appl. Spectrosc. 49:80-83). Detailed spectroscopic evidence is presented that at high pressure the equilibrium is shifted toward the 13-cis isomers and that the light to dark adaptation kinetics is accelerated. The change in isomeric composition with temperature and pressure as well as the kinetics support a two-state model activation volumes of -16 ml/mol for the transition of 13-cis to all-trans and -22 ml/mol for the reverse process. These compare with a conformational volume difference of 6.6 ml/mol, which may be attributed to the ionization of one or two residues or the formation of three hydrogen bonds.

Crystal Structure of the 13-cis Isomer of Bacteriorhodopsin in the Dark-adapted State

“…4,13 The isomeric composition in the dark-adapted state as well as in the light-adapted state is influenced by several factors, including the medium pH, 14,15 the aggregation state of the protein, 16 the hydration level, 17,18 and the hydraulic pressure. [19][20][21] Recent crystallographic studies of the trans photoreaction intermediates have revealed that the photo-isomerization of retinal from the all-trans to the 13-cis, 15-anti configuration is followed by a significant up-tilt of the C12-C14 region of retinal that is coupled with large displacements of residues (Leu93 and Trp182) contacting the C13 methyl group of retinal. 22,23 On the other hand, little information has been available as to how much these residues move during the dark adaptation, i.e.…”

Change in local dynamics of bacteriorhodopsin with retinal isomerization under pressure as studied by fast magic angle spinning NMR

“…Thus, the retinal isomeric ratio is insensitive to the variation in temperature, as has been reported in other studies. 15,16 Therefore, the increase in pressure is owing to fast-MAS-induced changes in dynamics at around Ala81 and Ala84, together with retinal isomerization from all-trans to 13-cis configurations. Figure 2 shows the 13 C CP-MAS NMR spectra of [3- 13 C]Ala-labeled bR at 4, 6, 8, 10 and 12 kHz.…”

“…Moreover, it was Bacteriorhodopsin and fast magic angle-spinning NMR I Kawamura et al confirmed by extraction techniques and absorption spectroscopy performed over a range of low to high pressure that the equilibrium constant of the all-trans to 13-cis, 15-syn isomerization decreased because of two processes that induce changes in molar volume in the dark. [15][16][17] As the population of 13-cis, 15-syn increased with pressure, the dynamics of the Ala81 and Ala84 residues in the vicinity of retinal increased the signal intensity at 16.6 p.p.m. Therefore, we concluded that the change in the dynamics of the residues was coupled with the change in molar volume.…”

“…13 C high-resolution solid-state NMR spectra were recorded on a Chemagnetics CMX-400 infinity FT-NMR spectrometer, operating at 100. 16 MHz for carbon nuclei, equipped with a 4.0-mm double resonance MAS probe. Cross polarization magic angle spinning (CP-MAS) with a variable amplitude contact pulse of observed nucleus and single-pulse excitation with dipolar decoupling and magic angle-spinning (DD-MAS) experiments were performed.…”

“…[12][13][14] The population of the 13-cis, 15-syn retinal in an inactive state increases under pressure with a negative change in molar volume, as reported by various groups. [15][16][17] Pressure effects on bR in the dark have suggested that the isomer equilibrium constant of the all-trans to 13-cis, 15-syn states decreases via two processes that induce different negative changes in molar volume upon the application of pressure. 17 The first process, which occurs at 10 MPa, may be caused by the change in the conformations of functionally important residues, such as Tyr185 and Asp212 in the vicinity of retinal.…”