The rhodopsin-lumirhodopsin transition has been investigated by Fourier transform infrared difference spectroscopy using isotope-labeled retinals. In the transition, two protonated carboxyl groups are involved. Another carbonyl band, located at 1725 cm-1 in rhodopsin, is shifted to 1731.5 cm-1 in lumirhodopsin. This line is tentatively assigned to a carbonyl stretching vibration of a peptide bond adjacent to the nitrogen of a proline residue. The C=N stretching vibration of rhodopsin could unequivocally be assigned to a band at 1659 cm-1. In contrast to rhodopsin and bathorhodopsin, the C=N stretching vibration of lumirhodopsin is at a low position, i.e., at 1635 cm-1, and exhibits only a downshift of 4 cm-1 upon deuteriation of the nitrogen. The C15-H rocking vibration of rhodopsin is assigned to the unusual high position of 1456 cm-1 and shifts into the normal region upon formation of lumirhodopsin. From these results, it is concluded that, whereas the environment of the Schiff base in rhodopsin, bathorhodopsin, and isorhodopsin is approximately the same, large changes occur with the formation of lumirhodopsin. From the assignment of the C10-C11 stretching vibration in bathorhodopsin and lumirhodopsin, a 10-s-cis geometry of lumirhodopsin can be excluded.
Time-resolved refractive index changes taking place during the photocycle of bacteriorhodopsin (BR) in purple membrane were studied by photothermal beam deflection (PBD) upon 8 ns pulse excitation. The PBD signal was monitored in the time range from several microseconds to 10 ms and separated into its various components originating from different physical effects (thermal, volume change, and absorbance-determined contributions).
The rate of regeneration of rhodopsin, from I I-cis-retinal and opsin, and bacteriorhodopsin from all-transretinal and bacterio-opsin, in the presence or absence of compounds whose structures partially resemble retinal were measured. Some of these compounds severely slowed down the regeneration process, but did not influence the extent of regeneration. In the case of compounds with a carbonyl functional group they were not joined to the active site of the apo-protein via a Schiffs base linkage since after treatment with NaBH4 an active apo-protein remained. The most effective inhibitors of rhodopsin regeneration were molecules whose structure could be superimposed on 9 4 or 11-cis retinal up to carbon atom 11. These C13 and CIS molecules were not distinguished between aldehyde, ketone or alcohol functional groups.The regeneration of bacteriorhodopsin was not inhibited by retinal analogues with short side chains. The most effective inhibitors were the all-trans C1 d d e h y d e (j-ionylideneacetaldehyde) or c 1 8 -ketone (/I-ionylidenepent-3-ene-2-one) which, compared to retinal, lack two or three carbon atoms from the end of the polyene chain. The inhibition was very dependent upon the presence of the all-trans isomer and required aldehyde or ketone as functional group; nitriles and alcohols were less effective. However, similarly to retinol, the all-trans CI7 and c 1 8 alcohols underwent a bathochromic shift and showed fine-structured spectra when mixed with bacterio-opsin
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