Rhodopsin experiments

In connection with the problem of oscillations discussed by previous speakers and other types of dynamical behavior of membranes, it would probably be timely to mention here in some more detail the experiments with vision rhodopsin that were performed in our institute by using the Mossbauer spectroscopy method [G. R. Kalamkarov et al., Doklady Biophys., 219, 126 (1974)]. These experiments manifested the existence of reversible photo-induced conformational changes in the photoreceptor membrane even at such low temperature as 77°K. We have labeled various samples of solubilized rhodopsin and of photoreceptor membranes by iron ascorbate enriched with Fe57 and looked for the change of Mossbauer spectra caused by the illumination of our samples. 

Chemical cross-linking experiments between rhodopsin and Gat indicate that residue 240 in IC 3 of rhodopsin is near the N- and C-termini of Ga, as well as the a4//J6 loop (Fig. 5 Cai et at., 2001 Itoh et at., 2001). Five residues at the C-terminus of Ga were shown to specifically interact with four noncontiguous residues in IC 3 (Liu et at., 1995). Peptides corresponding to IC 3 also cross-link to the N-terminus of Ga and the C-terminus of G/J (Taylor et at., 1994, 1996). As mentioned previously, the binding site for the C-terminal peptide of Gat was mapped to the inner face of helix VI (Janz and Farrens, 2004), and both the Ga and Gy C-termini of Gt interact with IC 4 of rhodopsin (Ernst et at., 2000). These data provide important information about specific contacts between receptors and G proteins however, more constraints are needed to improve our current model of the receptor-G protein interface. 

Several groups have demonstrated the use of trNOESY experiments to determine the structure of peptides bound to their receptor. For example, Kisselev et al. determined the 3D solution structure of the transductin a-subunit bound to light-activated rhodopsin (51). They found that the peptide IKENLKDCGLF formed an a-helix terminated by an open reverse turn (Fig. 11), while in contrast the conformation of the peptide remained disordered when in contact with nonactivated rhodopsin. Their findings led to the development of derivatives of the peptide that maintained the binding conformation and had improved affinity (73). Importantly, in their studies they used rhodopsin in a membrane environment (extracted from bovine rods), emphasizing the capabilities of the method for the study of integral membrane receptor directed inhibitors in their natural environment. 

Wald, et. al97,98. performed a set of experiments during the 1940 s that purported to demonstrate the formation of rhodopsin from either retinene, (now known as retinal) or Vitamin A, and a native protein. While their work involved materials showing a peak absorption at 500 nm, this is the wavelength of peak isotropic absorption of a large number of dipolar retinoids. Such a peak is not exclusive to the chromophoies of vision. Neither is it relevant to the anisotropic absorption spectrum of the chromophores of vision. 

They did not start from reagent grade chemicals. The experiments involved the in-vitro bleaching of native rhodopsin in solution. They proposed that they caused the disassociation of rhodopsin into a protein and retinaldehyde in accordance with the conventional wisdom of the time. This wisdom conflicted somewhat with the earlier ideas of Kuhne but became the basis of the current conventional wisdom. They claimed to have removed the native retinene, and then recombined colorless rhodopsin-protein, free of all native retinene, with synthetic retinene, in high concentration. This was accomplished by letting the mixture set in the dark. The pH of these solutions was not described as a function of time. They demonstrated that the material after setting in the dark for 60 minutes exhibited a rise in extinction coefficient of 2 1 over a similar sample without the added retinene,. Both samples exhibited a peak absorption near 500 nm following the experiments. It is not clear why the reference sample showed any absorption at 500 nm if it was truly free of all native retinene,. 

We now know the entire amino acid sequence of the putative rhodopsin. It is noteworthy that when these sequencing experiments were performed, there was no report of a residue containing a retinoid attached to an amino acid by a Schiff-base. No report could be found that even mentions a retinoid in the residue. 

Note carefully that the short wave peak in the curve for the dilute retinoids (alias rhodopsin), and labeled A, does not correspond in wavelength to the peak for any of the in-vitro materials. This is to be expected since the in-vitro experiments are usually performed with an alcohol solvent. Shifts in wavelength of 15-20 nm as a function of solvent are frequently encountered in in-vitro experiments146. 

The purpose of this review is to examine experimental information concerning the structure and function of the G protein-coupled serotonin receptors in the three-dimensional context provided by the structure of rhodopsin. A critical examination of the suitability of rhodopsin as a template for serotonin receptor modeling from the level of sequence alignment to interpretation of biochemical experiments of relevance to the issues of structure-function relationships is presented. 

Low temperature experiments have shown the formation of hypso intermediates from several species [99,103,105-107]. The study of early photoconversion processes in squid [108], which also involved the evaluation of the relative quantum yields among the four pigments (squid rhodopsin, squid batho-, hypso- and isorhodopsin) showed that hypsorhodopsin is a common intermediate of rhodopsin and isorhodopsin there is no direct conversion between rhodopsin and isorhodopsin bathorhodopsin is not converted directly to hypsorhodopsin and both rhodopsin and isorhodopsin convert more efficiently to bathorhodopsin than to hypsorhodopsin. While a temperature dependence of the relaxation processes from the excited state of rhodopsin, and an assumption that batho could be formed from one of the high vibrational levels of the ground state hypso have been invoked to explain these findings [108], the final clarification of this matter awaits results from subpicosecond laser photolysis experiments at liquid helium temperature. 

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