Determination of retinal chromophore structure in bacteriorhodopsin with resonance Raman spectroscopy

Abstract

The analysis of the vibrational spectrum of the retinal chromophore in bacteriorhodopsin with isotopic derivatives provides a powerful "structural dictionary" for the translation of vibrational frequencies and intensities into structural information. Of importance for the proton-pumping mechanism is the unambiguous determination of the configuration about the C13=C14 and C=N bonds, and the protonation state of the Schiff base nitrogen. Vibrational studies have shown that in light-adapted BR568 the Schiff base nitrogen is protonated and both the C13=C14 and C=N bonds are in a trans geometry. The formation of K625 involves the photochemical isomerization about only the C13=C14 bond which displaces the Schiff base proton into a different protein environment. Subsequent Schiff base deprotonation produces the M412 intermediate. Thermal reisomerization of the C13=C14 bond and reprotonation of the Schiff base occur in the M412------O640 transition, resetting the proton-pumping mechanism. The vibrational spectra can also be used to examine the conformation about the C--C single bonds. The frequency of the C14--C15 stretching vibration in BR568, K625, L550 and O640 argues that the C14--C15 conformation in these intermediates is s-trans. Conformational distortions of the chromophore have been identified in K625 and O640 through the observation of intense hydrogen out-of-plane wagging vibrations in the Raman spectra (see Fig. 2). These two intermediates are the direct products of chromophore isomerization. Thus it appears that following isomerization in a tight protein binding pocket, the chromophore cannot easily relax to a planar geometry. The analogous observation of intense hydrogen out-of-plane modes in the primary photoproduct in vision (Eyring et al., 1982) suggests that this may be a general phenomenon in protein-bound isomerizations. Future resonance Raman studies should provide even more details on how bacterio-opsin and retinal act in concert to produce an efficient light-energy convertor. Important unresolved questions involve the mechanism by which the protein catalyzes deprotonation of the L550 intermediate and the mechanism of the thermal conversion of M412 back to BR568. Also, it has been shown that under conditions of high ionic strength and/or low light intensity two protons are pumped per photocycle (Kuschmitz & Hess, 1981). How might this be accomplished?(ABSTRACT TRUNCATED AT 400 WORDS)

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

“…This process required breaking the Schiff base linker by subjecting the protein to EtOH followed by extraction of retinals into hexanes and HPLC purification (55). For bacteriorhodopsin, the results obtained in this way (56) agree well with data from other methods (26,27). In other cases, however, this protein treatment led to less consistent results.…”

“…1B (14,40). According to this model, blue light excitation leads to a retinal all-trans to 13-cis isomerization, resulting in a red-shifted first intermediate P 500 1 (12) resembling a K-like state, which most likely contains a 13-cis,15-anti retinal Schiff base chromophore similar to Bacteriorhodopsin (27). To our knowledge, such red-shifted K-like intermediates occur in all microbial retinal proteins (38).…”

“…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).…”

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

“…In addition, the relative orientation and interaction of the retinal chromophore with the F-helix is supported by studies,using a photosensitive analog of retinal [14]. The chromophore was fixed in all-trans configuration in agreement with resonance Raman studies [15]. 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.…”

Sensory rhodopsin I photocycle intermediate SRI380 contains 13‐cis retinal bound via an unprotonated Schiff base

“…In the HtrI-SRI complex the photocycle rates are independent of pH in the range of 3 to 8. In contrast, the decay time of the SRIsso intermediate of uncomplexed SRI shows an exponential increase with increasing pH [5] accompanied by a 40 nm blue shift of the initial state absorption maximum to 550 nm at alkaline PH [4,61. BR-M, absorbing maximally at 410 nm, is a key intermediate in the photocycle of BR and the retinal was shown by resonance Raman measurements to be bound to the protein via a deprotonated Schiffbase in its 13-c@ 14s"trans, 15"anti configuration [7,8]. Continuous illumination of HR samples in the presence of azide leads to accumulation of HR:,, [9] which shows a Resonance Raman spectrum [lo] similar to BR-M.…”