Fourier-transform infrared difference spectroscopy of rhodopsin and its photoproducts at low temperature

Abstract

Fourier-transform infrared difference spectroscopy has been used to detect the vibrational modes in the chromophore and protein that change in position or intensity between rhodopsin and the photoproducts formed at low temperature (70 K), bathorhodopsin and isorhodopsin. A method has been developed to obtain infrared difference spectra between rhodopsin and bathorhodopsin, bathorhodopsin and isorhodopsin, and rhodopsin and isorhodopsin. To aid in the identification of the vibrational modes, we performed experiments on deuterated and hydrated films of native rod outer segments and rod outer segments regenerated with either retinal containing 13C at carbon 15 or 15-deuterioretinal. Our infrared measurements provide independent verification of the resonance Raman result that the retinal in bathorhodopsin is distorted all-trans. The positions of the C = N stretch in the deuterated pigment and the deuterated pigments regenerated with 11-cis-15-deuterioretinal or 11-cis-retinal containing 13C at carbon 15 are indicative that the Schiff-base linkage is protonated in rhodopsin, bathorhodopsin, and isorhodopsin. Furthermore, the C = N stretching frequency occurs at the same position in all three species. The data indicate that the protonated Schiff base has a C = N trans conformation in all three species. Finally, we present evidence that, even in these early stages of the rhodopsin photosequence, changes are occurring in the opsin and perhaps the associated lipids.

The photochemical event in rhodopsins

“…So did the v(C=N+) band between Rh, B, and isorhodopsin. Most significantly, however, the three Ndeuterated samples gave three different v(C=N+) frequencies (167). They are 1624 for B, 1631 for isorhodopsin, and 1624 cm-for Rh (164,200) in agreement with previous results of Siebert et al (164,200).…”

“…Now, since this is so, the uniform v(C=Nf ) might not correspond to the same potential, average proton position and charge distribution in the proton bridge. This is clearly illustrated by the recent differential infrared experiments of Bagley et al (167). These researchers performed experiments on hydrated and deuterated films of native rod outer segment (ROS) and ROS regenerated with retinals with isotopic substitution at C-15.…”

Infrared-Detectable Group Senses Changes in Charge Density on the Nickel Center in Hydrogenase from Chromatium vinosum

“…Such bands are absent from the low-temperature spectra of myoglobin (Alben & Caughey, 1968), cytochrome oxidase (Fiamingo et al, 1981), rhodopsin (Bagley et al, 1985(Bagley et al, , 1989, and bacteriorhodopsin (Bagley et al, 1982). Such bands are also absent from the infrared spectra of another Ni,Fe enzyme, CO dehydrogenase (Kumar & Ragsdale, 1992).…”

The early development and application of FTIR difference spectroscopy to membrane proteins: A personal perspective

“…They emphasized the potential of IR-difference spectroscopy to study protein conformational changes. Along with our initial work in 1981, Laura Eisenstein at the University of Illinois used FTIR difference spectroscopy starting in 1982 to study BR [16,17] and later rhodopsin [18,19]. In the case of BR, this work confirmed that the SB in the BR 570 and the K 630 intermediate are both protonated.…”

“…In one experiment reported in 1981, lysines in BR were substituted with lysines where the ε-amino group was either completely or partial labeled with 15 N [6]. The results disproved the previously suggested [140] hypothesis [18] that two different lysine residues interacted with the retinylidene Schiff base (SB). In a second experiment ε-amino N 15 lysine labeling was combined with proteolytic fragmentation and recombination of intact BR from these fragments [194].…”