Cyclopropyl and isopropyl derivatives of 11-cis and 9-cis retinals at C-9 and C-13: subtle steric differences with major effects on ligand efficacy in rhodopsin.

Retinal is the natural ligand (chromophore) of the vertebrate rod visual pigment. It occurs in either the 11-cis (rhodopsin) or the 9-cis (isorhodopsin) configuration. In its evolution to a G protein coupled photoreceptor, rhodopsin has acquired exceptional photochemical properties.

Cell-free expression and labeling strategies for a new decade in solid-state NMR.

Although solid-state NMR and cell-free expression have recently become standard methods in biology, the combination of the two is still at a very early stage of development. In this article, we will explore several approaches by which cell-free expression could help solid-state NMR in its quest for biomolecular structure and mechanism elucidation. Far from being just another structure determination technique, this quest is motivated by the unique possibility of using solid-state NMR to determine the high resolution structure of a membrane protein within its native environment, the lipid membrane.

Structural study of the membrane protein MscL using cell-free expression and solid-state NMR.

High-resolution structures of membrane proteins have so far been obtained mostly by X-ray crystallography, on samples where the protein is surrounded by detergent. Recent developments of solid-state NMR have opened the way to a new approach for the study of integral membrane proteins inside a membrane. At the same time, the extension of cell-free expression to the production of membrane proteins allows for the production of proteins tailor made for NMR.

Methyl substituents at the 11 or 12 position of retinal profoundly and differentially affect photochemistry and signalling activity of rhodopsin.

The C-11=C-12 double bond of the retinylidene chromophore of rhodopsin holds a central position in its light-induced photoisomerization and hence the photosensory function of this visual pigment. To probe the local environment of the HC-11=C-12H element we have prepared the 11-methyl and 12-methyl derivatives of 11-Z retinal and incorporated these into opsin to generate the rhodopsin analogs 11-methyl and 12-methyl rhodopsin. These analog pigments form with much slower kinetics and lower efficiency than the native pigment.

Accurate measurements of 13C-13C J-couplings in the rhodopsin chromophore by double-quantum solid-state NMR spectroscopy.

A new double-quantum solid-state NMR pulse sequence is presented and used to measure one-bond 13C-13C J-couplings in a set of 13C2-labeled rhodopsin isotopomers. The measured J-couplings reveal a perturbation of the electronic structure at the terminus of the conjugated chain but show no evidence for protein-induced electronic perturbation near the C11-C12 isomerization site. This work establishes NMR methodology for measuring accurate 1JCC values in noncrystalline macromolecules and shows that the measured J-couplings may reveal local electronic perturbations of mechanistic significance.

The ring of the rhodopsin chromophore in a hydrophobic activation switch within the binding pocket.

The current view that the beta-ionone ring of the rhodopsin chromophore vacates its binding pocket within the protein early in the photocascade has been adopted in efforts to provide structural models of photoreceptor activation. This event casts doubt on the ability of this covalently bonded ligand to participate directly in later stages involving activation of the photoreceptor and it is difficult to translate into predictions for the activation of related G protein-coupled receptors by diffusable ligands (e.g.

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.

Conformational similarities in the beta-ionone ring region of the rhodopsin chromophore in its ground state and after photoactivation to the metarhodopsin-I intermediate.

High-resolution solid-state NMR methods have been used to analyze the conformation of the chromophore in the late photointermediate metarhodopsin-I, from observation of (13)C nuclei introduced into the beta-ionone ring (at the C16, C17, and C18 methyl groups) and into the adjoining segment of the polyene chain (at C8). Bovine rhodopsin in its native membrane was also regenerated with retinal that was (13)C-labeled close to the 11-Z bond (C20 methyl group) to provide a reporter for optimizing and quantifying the photoconversion to metarhodopsin-I. Indirect photoconversion via the primary intermediate, bathorhodopin, was adopted as the preferred method since approximately 44% conversion to the metarhodopsin-I component could be achieved, with only low levels (approximately 18%) of ground-state rhodopsin remaining.

Extraction of carotenoids from feces, enabling the bioavailability of beta-carotene to be studied in Indonesian children.

Previously, we have presented a method for quantifying beta-carotene bioavailability based on analysis in serum, following administration of (13)C-labeled beta-carotene. Because stool samples can be collected noninvasively, we have now extended the method to measure the bioavailability based on measurements in feces. An extraction method was developed to enable measurement of concentrations and degree of isotopic enrichment of retinol, retinyl palmitate and carotenoids in feces.

Relative orientation between the beta-ionone ring and the polyene chain for the chromophore of rhodopsin in native membranes.

Rotational resonance solid state nuclear magnetic resonance has been used to determine the relative orientation of the beta-ionone ring and the polyene chain of the chromophore 11-Z-retinylidene of rhodopsin in rod outer segment membranes from bovine retina. The bleached protein was regenerated with either 11-Z-[8,18-(13)C(2)]retinal or 11-Z-[8,16/17(13)C(2)]retinal, the latter having only one (13)C label at either of the chemically equivalent positions 16 and 17. Observation of (13)C selectively enriched in the ring methyl groups, C16/17, revealed alternative conformational states for the ring.

Time-resolved resonance Raman analysis of chromophore structural changes in the formation and decay of rhodopsin's BSI intermediate.

Time-resolved resonance Raman microchip flow experiments are performed to obtain the vibrational spectrum of the chromophore in rhodopsin's BSI intermediate and to probe structural changes in the bathorhodopsin-to-BSI and BSI-to-lumirhodopsin transitions. Kinetic Raman spectra from 250 ns to 3 micros identify the key vibrational features of BSI. BSI exhibits relatively intense HOOP modes at 886 and 945 cm(-1) that are assigned to C(14)H and C(11)H=C(12)H A(u) wags, respectively.

Interaction of internal water molecules with the schiff base in the L intermediate of the bacteriorhodopsin photocycle.

In the photocycle of bacteriorhodopsin (BR), the first proton movement, from the Schiff base to Asp85, occurs after the formation of the L intermediate. In L, the C [double bond] N bond of the Schiff base is strained, and the nitrogen interacts strongly with its counterion. The present study seeks to detect the interaction of internal water molecules with the Schiff base in L using difference FTIR spectroscopy at 170 K.