Dark-adapted bacteriorhodopsin contains 13-cis, 15-syn and all-trans, 15-anti retinal Schiff bases.

Abstract

ABSTRACT13C NMR spectra of Iyophilized dark-adapted [14-13C]retinyl-labeled bacteriorhodopsin show a large anomalous upfield shift for the 13C-14 resonance assigned to the 13-cis isomer, relative to both the all-trans isomer and model compounds. We attribute this to the so-called y effect, which results from a steric interaction between the C-14 retinal proton and the protons on the e CH2 of the lysine. As a consequence of this observation, we infer that dark-adapted bacteriorhodopsin is composed of a mixture of all-trans,15-anti (trans or E) and 13-cis,15-syn (cis or Z) isomers. These occur in an approximate 4:6 ratio and are commonly identified as bRm and bR54. This conclusion is based on an examination of the isotropic and anisotropic chemical shifts and a comparison with 13C shifts of the carbons adjacent to the C=N linkage in protonated ketinines. Other possible origins for the anomalous shift are examined and shown to be insufficient to account for either the size of the shift or the nature of the shift tensor. We discuss the consequences of this finding for the structure and photochemistry of bacteriorhodopsin.Bacteriorhodopsin (bR), the single protein of the purple membrane (PM) of Halobacterium halobium (1), has been the subject of considerable experimental scrutiny for some time. Like rhodopsin (2), it contains as its chromophore the polyene aldehyde retinal, connected via a Schiff base linkage to the E-amino group of a lysine side chain (3). Although bR appears to resemble visual pigments (4) (14), the configuration of the C=-N Schiff base linkage in bR has not been considered. In addition, the interesting possibility that isomerization about this bond might occur during the photocycle has been largely overlooked. The inattention to this important question can be attributed in part to the absence of an experimental means to discriminate definitively between syn and anti isomers in bR. In a recent paper (15) we demonstrated that high-resolution solid-state 3C NMR is a potent means of establishing configuration about C=C bonds in bR. Here, we present evidence that it is equally effective in determining the C=N bond configuration and evinces that dark-adapted bR contains alltrans,15-anti and 13-cis,15-syn isomers in an approximately 4:6 ratio. The implications of this finding for both the structure of the chromophore and the bR photocycle will also be discussed. MATERIALS AND METHODS13C-14-labeled retinal was prepared by the method of ref. 16 and incorporated into white membrane as described (8, 15). The reconstituted PM was then lyophilized at 0.1 mm Hg (1 mm Hg = 133 Pa) and packed into a Kel-F rotor of the Andrew-Beams design (17). 13C magic-angle sample spinning (MASS) spectra were obtained at various spinning frequencies between 1.9 and 3.2 kHz, with a 13C frequency of 79.9 MHz. Typically, (,)1/21r) = 50 kHz was used for cross-polarization. Subsequently, the magnetization was sampled in the presence of 1H decoupling fields of 125 kHz. Usually, 15,000 transients were accumulated, with a recycle de...

Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy

“…2B). The C14 chemical shift is very sensitive to the conformation of the chromophore (26), and the value of 126.3 ppm is, therefore, a very strong indicator for an all-trans,15-anti chromophore conformation as observed for bacteriorhodopsin and proteorhodopsin (SI Appendix, Table S1). By contrast, the C14 signal of the 13-cis,15-syn chromophore in dark-adapted bacteriorhodopsin appears at 111 ppm.…”

“…Without any illumination, microbial retinal proteins thermally equilibrate into a dark state (25). In the case of bacteriorhodopsin, for example, this state contains a mixture of two species termed bacteriorhodopsin 568 (all-trans,15-anti retinal Schiff base) and bacteriorhodopsin 548 (13-cis,15-syn conformation) (26,27). On illumination, light adaption occurs from the dark state to the ground state, which contains only the all-trans,15-anti conformer as the photocycle starting point (28).…”

Cellular solid-state nuclear magnetic resonance spectroscopy

“…6 for a recent review). Indeed, ssNMR has already been used to study individual molecular components in the context of natural bilayers (7,8), bacterial cell walls (9), and cellular organelles (10).Here, we introduce a general approach to investigate structure and dynamics of an arbitrary molecular target and its potential molecular partners in a cellular setting. Our studies focuses on the Gram-negative bacterial cell that is characterized by a molecularly complex but architecturally unique envelope, consisting of two lipid bilayers, the inner and outer membrane (IM, OM), separated by the periplasm containing the peptidoglycan (PG) layer (Fig.…”

“…6 for a recent review). Indeed, ssNMR has already been used to study individual molecular components in the context of natural bilayers (7,8), bacterial cell walls (9), and cellular organelles (10).…”

Quantum Chemistry of in situ Retinal: Study of the Spectral Properties and Dark Adaptation of Bacteriorhodopsin

“…The protein containing the 13-cis retinal isomer of bRis referred to as the dark adapted (DA) pigment of bR, bR 548 , which absorbs at 548 nm. bR 548 contains, actually, retinal in a 13-cis ,15-syn geometry as 3 suggested first in [15] and observed in [16,17,18]. bR 548 undergoes a light-driven photocycle similar to that of all-trans retinal which also involves an initial photoisomerization about the C 13 -C 14 double bond (see Fig.…”