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J Courtin

Publications and source records attributed to J Courtin.

18 recordsLinked to original sources
PubMed1991

13C magic-angle spinning NMR studies of bathorhodopsin, the primary photoproduct of rhodopsin.

Magic-angle spinning NMR spectra have been obtained of the bathorhodopsin photointermediate trapped at low temperature (less than 130 K) by using isorhodopsin samples regenerated with retinal specifically 13C-labeled at positions 8, 10, 11, 12, 13, 14, and 15. Comparison of the chemical shifts of the bathorhodopsin resonances with those of an all-trans-retinal protonated Schiff base (PSB) chloride salt show the largest difference (6.2 ppm) at position 13 of the protein-bound retinal. Small differences in chemical shift between bathorhodopsin and the all-trans PSB model compound are also observed at positions 10, 11, and 12. The effects are almost equal in magnitude to those previously observed in rhodopsin and isorhodopsin. Consequently, the energy stored in the primary photoproduct bathorhodopsin does not give rise to any substantial change in the average electron density at the labeled positions. The data indicate that the electronic and structural properties of the protein environment are similar to those in rhodopsin and isorhodopsin. In particular, a previously proposed perturbation near position 13 of the retinal appears not to change its position significantly with respect to the chromophore upon isomerization. The data effectively exclude charge separation between the chromophore and a protein residue as the main mechanism for energy storage in the primary photoproduct and argue that the light energy is stored in the form of distortions of the bathorhodopsin chromophore.

Chlorides↗
PubMed1990

Solid-state NMR studies of the mechanism of the opsin shift in the visual pigment rhodopsin.

Solid-state 13C NMR spectra have been obtained of bovine rhodopsin and isorhodopsin regenerated with retinal selectively 13C labeled along the polyene chain. In rhodopsin, the chemical shifts for 13C-5, 13C-6, 13C-7, 13C-14, and 13C-15 correspond closely to the chemical shifts observed in the 11-cis protonated Schiff base (PSB) model compound. Differences in chemical shift relative to the 11-cis PSB chloride salt are observed for positions 8 through 13, with the largest deshielding (6.2 ppm) localized at position 13. The localized deshielding at C-13 supports previous models of the opsin shift in rhodopsin that place a protein perturbation in the vicinity of position 13. Spectra obtained of isorhodopsin regenerated with 13C-labeled 9-cis-retinals reveal large perturbations at 13C-7 and 13C-13. The similar deshielding of the 13C-13 resonance in both pigments supports the presence of a protein perturbation near position 13. However, the chemical shifts at positions 7 and 12 in isorhodopsin are not analogous to those observed in rhodopsin and suggest that the binding site interactions near these positions are different for the two pigments. The implications of these results for the mechanism of the opsin shift in these proteins are discussed.

Animals↗
PubMed1990

Solid-state 13C and 15N NMR study of the low pH forms of bacteriorhodopsin.

The visible absorption of bacteriorhodopsin (bR) is highly sensitive to pH, the maximum shifting from 568 nm (pH 7) to approximately 600 nm (pH 2) and back to 565 nm (pH 0) as the pH is decreased further with HCl. Blue membrane (lambda max greater than 600 nm) is also formed by deionization of neutral purple membrane suspensions. Low-temperature, magic angle spinning 13C and 15N NMR was used to investigate the transitions to the blue and acid purple states. The 15N NMR studies involved [epsilon-15N]lysine bR, allowing a detailed investigation of effects at the Schiff base nitrogen. The 15N resonance shifts approximately 16 ppm upfield in the neutral purple to blue transition and returns to its original value in the blue to acid purple transition. Thus, the 15N shift correlates directly with the color changes, suggesting an important contribution of the Schiff base counterion to the "opsin shift". The results indicate weaker hydrogen bonding in the blue form than in the two purple forms and permit a determination of the contribution of the weak hydrogen bonding to the opsin shift at a neutral pH of approximately 2000 cm-1. An explanation of the mechanism of the purple to blue to purple transition is given in terms of the complex counterion model. The 13C NMR experiments were performed on samples specifically 13C labeled at the C-5, C-12, C-13, C-14, or C-15 positions in the retinylidene chromophore. The effects of the purple to blue to purple transitions on the isotropic chemical shifts for the various 13C resonances are relatively small. It appears that bR600 consists of at least four different species. The data confirm the presence of 13-cis- and all-trans-retinal in the blue form, as in neutral purple dark-adapted bR. All spectra of the blue and acid purple bR show substantial inhomogeneous broadening which indicates additional irregular distortions of the protein lattice. The amount of distortion correlates with the variation of the pH, and not with the color change.

Bacteriorhodopsins↗
PubMed1989

Solid-state 13C NMR of the retinal chromophore in photointermediates of bacteriorhodopsin: characterization of two forms of M.

Solid-state 13C NMR spectra of the M photocycle intermediate of bacteriorhodopsin (bR) have been obtained from purple membrane regenerated with retinal specifically 13C labeled at positions 5, 12, 13, 14, and 15. The M intermediate was trapped at -40 degrees C and pH = 9.5-10.0 in either 100 mM NaCl [M (NaCl)] or 500 mM guanidine hydrochloride [M (Gdn-HCl)]. The 13C-12 chemical shift at 125.8 ppm in M (NaCl) and 128.1 ppm in M (Gdn-HCl) indicates that the C13 = C14 double bond has a cis configuration, while the 13C-13 chemical shift at 146.7 ppm in M (NaCl) and 145.7 ppm in M (Gdn-HCl) demonstrates that the Schiff base is unprotonated. The principal values of the chemical shift tensor of the 13C-5 resonance in both M (NaCl) and M (Gdn-HCl) are consistent with a 6-s-trans structure and a negative protein charge localized near C-5 as was observed in dark-adapted bR. The approximately 5 ppm upfield shift of the 13C-5 M resonance (approximately 140 ppm) relative to 13C-5 bR568 and bR548 (approximately 145 ppm) is attributed to an unprotonated Schiff base in the M chromophore. Of particular interest in this study were the results obtained from 13C-14 M. In M (NaCl), a dramatic upfield shift was observed for the 13C-14 resonance (115.2 ppm) relative to unprotonated Schiff base model compounds (approximately 128 ppm). In contrast, in M (Gdn-HCl) the 13C-14 resonance was observed at 125.7 ppm. The different 13C-14 chemical shifts in these two M preparations may be explained by different C = N configurations of the retinal-lysine Schiff base linkage, namely, syn in NaCl and anti in guanidine hydrochloride.

Bacteriorhodopsins↗
PubMed1987

Low-temperature solid-state 13C NMR studies of the retinal chromophore in rhodopsin.

Magic angle sample spinning (MASS) 13C NMR spectra have been obtained of bovine rhodopsin regenerated with retinal prosthetic groups isotopically enriched with 13C at C-5 and C-14. In order to observe the 13C retinal chromophore resonances, it was necessary to employ low temperatures (-15-----35 degrees C) to restrict rotational diffusion of the protein. The isotropic chemical shift and principal values of the chemical shift tensor of the 13C-5 label indicate that the retinal chromophore is in the twisted 6-s-cis conformation in rhodopsin, in contrast to the planar 6-s-trans conformation found in bacteriorhodopsin. The 13C-14 isotropic shift and shift tensor principal values show that the Schiff base C = N bond is anti. Furthermore, the 13C-14 chemical shift (121.2 ppm) is within the range of values (120-123 ppm) exhibited by protonated (C = N anti) Schiff base model compounds, indicating that the C = N linkage is protonated. Our results are discussed with regard to the mechanism of wavelength regulation in rhodopsin.

Animals↗