Towards an interpretation of 13C chemical shifts in bathorhodopsin, a functional intermediate of a G-protein coupled receptor.

Photoisomerization of the membrane-bound light receptor protein rhodopsin leads to an energy-rich photostate called bathorhodopsin, which may be trapped at temperatures of 120 K or lower. We recently studied bathorhodopsin by low-temperature solid-state NMR, using in situ illumination of the sample in a purpose-built NMR probe. In this way we acquired (13)C chemical shifts along the retinylidene chain of the chromophore. Here we compare these results with the chemical shifts of the dark state chromophore in rhodopsin, as well as with the chemical shifts of retinylidene model compounds in solution. An earlier solid-state NMR study of bathorhodopsin found only small changes in the (13)C chemical shifts upon isomerization, suggesting only minor perturbations of the electronic structure in the isomerized retinylidene chain. This is at variance with our recent measurements which show much larger perturbations of the (13)C chemical shifts. Here we present a tentative interpretation of our NMR results involving an increased charge delocalization inside the polyene chain of the bathorhodopsin chromophore. Our results suggest that the bathochromic shift of bathorhodopsin is due to modified electrostatic interactions between the chromophore and the binding pocket, whereas both electrostatic interactions and torsional strain are involved in the energy storage mechanism of bathorhodopsin.

Double-quantum 13C nuclear magnetic resonance of bathorhodopsin, the first photointermediate in mammalian vision.

The 13C chemical shifts of the primary visual photointermediate bathorhodopsin have been observed by performing double-quantum magic-angle-spinning NMR at low temperature in the presence of illumination. Strong isomerization shifts have been observed upon the conversion of rhodopsin into bathorhodopsin.

Effect of a combination of isoflavones and Actaea racemosa Linnaeus on climacteric symptoms in healthy symptomatic perimenopausal women: a 12-week randomized, placebo-controlled, double-blind study.

OBJECTIVE: To investigate the effects of a novel dietary supplement containing soy isoflavones and Actaea racemosa Linnaeus (formerly called Cimicifuga racemosa L.) on climacteric symptoms in healthy perimenopausal women. DESIGN: In a multicenter, randomized, placebo-controlled, double-blind study, 124 women experiencing at least five vasomotor symptoms every 24 hours were randomized to receive daily either a phytoestrogen-containing supplement (n = 60) or placebo (n = 64) for 12 weeks. The modified Kupperman Index and Greene Climacteric Scale, a visual analogue scale designed to measure quality of life and the daily number and severity of hot flushes, was used in the screening period and in weeks 6 and 12. Changes in these scores from baseline were calculated. RESULTS: At weeks 6 and 12, all scores in both groups had improved compared with baseline, though the overall difference in scores between the groups was not statistically significant. CONCLUSION: The supplement containing soy isoflavones and A racemosa L. had no statistically significant effect on climacteric symptoms in perimenopausal women experiencing at least five vasomotor symptoms per day.

Protein-induced bonding perturbation of the rhodopsin chromophore detected by double-quantum solid-state NMR.

We have obtained carbon-carbon bond length data for the functional retinylidene chromophore of rhodopsin, with a spatial resolution of 3 pm. The very high resolution was obtained by performing double-quantum solid-state NMR on a set of noncrystalline isotopically labelled bovine rhodopsin samples. We detected localized perturbations of the carbon-carbon bond lengths of the retinylidene chromophore. The observations are consistent with a model in which the positive charge of the protonated Schiff base penetrates into the polyene chain and partially concentrates around the C13 position. This coincides with the proximity of a water molecule located between the glutamate-181 and serine-186 residues of the second extracellular loop, which is folded back into the transmembrane region. These measurements support the hypothesis that the polar residues of the second extracellular loop and the associated water molecule assist the rapid selective photoisomerization of the retinylidene chromophore by stabilizing a partial positive charge in the center of the polyene chain.

Control of the pump cycle in bacteriorhodopsin: mechanisms elucidated by solid-state NMR of the D85N mutant.

By varying the pH, the D85N mutant of bacteriorhodopsin provides models for several photocycle intermediates of the wild-type protein in which D85 is protonated. At pH 10.8, NMR spectra of [zeta-(15)N]lys-, [12-(13)C]retinal-, and [14,15-(13)C]retinal-labeled D85N samples indicate a deprotonated, 13-cis,15-anti chromophore. On the other hand, at neutral pH, the NMR spectra of D85N show a mixture of protonated Schiff base species similar to that seen in the wild-type protein at low pH, and more complex than the two-state mixture of 13-cis,15-syn, and all-trans isomers found in the dark-adapted wild-type protein. These results lead to several conclusions. First, the reversible titration of order in the D85N chromophore indicates that electrostatic interactions have a major influence on events in the active site. More specifically, whereas a straight chromophore is preferred when the Schiff base and residue 85 are oppositely charged, a bent chromophore is found when both the Schiff base and residue 85 are electrically neutral, even in the dark. Thus a "bent" binding pocket is formed without photoisomerization of the chromophore. On the other hand, when photoisomerization from the straight all-trans,15-anti configuration to the bent 13-cis,15-anti does occur, reciprocal thermodynamic linkage dictates that neutralization of the SB and D85 (by proton transfer from the former to the latter) will result. Second, the similarity between the chromophore chemical shifts in D85N at alkaline pH and those found previously in the M(n) intermediate of the wild-type protein indicate that the latter has a thoroughly relaxed chromophore like the subsequent N intermediate. By comparison, indications of L-like distortion are found for the chromophore of the M(o) state. Thus, chromophore strain is released in the M(o)-->M(n) transition, probably coincident with, and perhaps instrumental to, the change in the connectivity of the Schiff base from the extracellular side of the membrane to the cytoplasmic side. Because the nitrogen chemical shifts of the Schiff base indicate interaction with a hydrogen-bond donor in both M states, it is possible that a water molecule travels with the Schiff base as it switches connectivity. If so, the protein is acting as an inward-driven hydroxyl pump (analogous to halorhodopsin) rather than an outward-driven proton pump. Third, the presence of a significant C [double bond] N syn component in D85N at neutral pH suggests that rapid deprotonation of D85 is necessary at the end of the wild-type photocycle to avoid the generation of nonfunctional C [double bond] N syn species.