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. Illumination isomerizes the chromophore to the all-trans isomer, which acts as a full agonist. This process is extremely efficient, and there is abundant evidence that the C-9 and C-13 methyl groups of retinal play a pivotal role in this process. To examine the steric limits of the C-9 and C-13 methyl binding pocket of the binding site, we have prepared C-9 and C-13 cyclopropyl and isopropyl derivatives of its native ligands and of α-retinal at C-9. Most isopropyl analogues show very poor binding, except for 9-cis-13-isopropylretinal. Most cyclopropyl derivatives exhibit intermediate binding activity, except for 9-cis-13-cyclopropylretinal, which presents good binding activity. In general, the binding site shows preference for the 9-cis analogues over the 11-cis analogues. In fact, 13-isopropyl-9-cis-retinal acts as a superagonist after illumination. Another surprising finding was that 9-cyclopropylisorhodopsin is more like native rhodopsin with respect to spectral and photochemical properties, whereas 9-cyclopropylrhodopsin behaves more like native isorhodopsin in these aspects.

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. We will examine the specific sample preparation requirements that such a goal imposes and how cell-free expression can play a key role in such a protocol.

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. We present here an in situ solid-state NMR study of a membrane protein selectively labeled through the use of cell-free expression. The sample consists of MscL (mechano-sensitive channel of large conductance), a 75kDa pentameric alpha-helical ion channel from Escherichia coli, reconstituted in a hydrated lipid bilayer. Compared to a uniformly labeled protein sample, the spectral crowding is greatly reduced in the cell-free expressed protein sample. This approach may be a decisive step required for spectral assignment and structure determination of membrane proteins by solid-state 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. The initial photochemistry and the signaling activity of the analog pigments were investigated by UV-vis and FTIR spectroscopy, and by a G protein activation assay. Our data indicate that the ultrafast formation of the first photointermediate is strongly perturbed by the presence of an 11-methyl substituent, but much less by a 12-methyl substituent. These results support the current concept of the mechanism of the primary photoisomerization event in rhodopsin. An important stronghold of this concept is an out-of-plane movement of the C-12H element, which is facilitated by torsion as well as extended positive charge delocalization into the C-10-C-13 segment of the chromophore. We argue that this mechanism is maintained principally with a methyl substituent at C-12. In addition, we show that both an 11-methyl and a 12-methyl substitutent perturb the photointermediate cascade and finally yield a low-activity state of the receptor. The 11-methyl pigment retains about 30% of the G protein activation rate of native rhodopsin, while the 12-methyl chromophore behaves like an inverse agonist up to at least 20 degrees C, trapping the protein in a perturbed Meta-I-like conformation. We conclude that the isomerization region of the chromophore and the spatial structure of the binding site are finely tuned, in order to achieve a high photosensory potential with an efficient pathway to a high-activity state.

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. neurotransmitters). The binding pocket fixes the formally equivalent pair of ring methyl groups (C16/C17) in different orientations that can be distinguished easily by (13)C NMR. Solid-state NMR observations on C16 and C17 are reported here that show instead that the ring is retained with strong selective interactions within the binding site into the activated state. We further show how increased steric interactions for this segment in the activated receptor can be explained by adjustment in the protein structure around the ring whilst it remains in its original location. This describes a plausible role for the ring in operating a hydrophobic switch from within the aromatic cluster of helix 6 of rhodopsin, which is coupled to electronic changes within the receptor through water-mediated, hydrogen-bonded networks between the conserved residues in G protein-coupled receptors.

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.

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. The additional photoproduct, isorhodopsin, was resolved in (13)C spectra from C8 in the chain, at levels of approximately 38%, and was shown using rotational resonance NMR to adopt the 6-s-cis conformation between the ring and the polyene chain. The C8 resonance was not shifted in the metarhodopsin-I spectral component but was strongly broadened, revealing that the local conformation had become less well defined in this segment of the chain. This line broadening slowed rotational resonance exchange with the C17 and C18 ring methyl groups but was accounted for to show that, despite the chain being more relaxed in metarhodopsin-I, its average conformation with respect to the ring was similar to that in the ground state protein. Conformational restraints are also retained for the C16 and C17 methyl groups on photoactivation, which, together with the largely preserved conformation in the chain, argues convincingly that the ring remains with strong contacts in its binding pocket prior to activation of the receptor. Previous conclusions based on photocrosslinking studies are considered in view of the current findings.

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 bioavailability of beta-carotene from pumpkin (n = 6) was found to be 1.8 times (interval, 0.6, 5.5) greater than that from spinach (n = 8), based on data from feces compared with 1.7 times (interval, 0.9, 3.1) based on data from serum.

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. Minor spectral components comprised around 26% of the chromophore. The major conformation (approximately 74%) has the chemical shift resolution required for measuring internuclear distances to (13)C in the retinal chain (C8) separately from each of these methyl groups. The resulting distance constraints, C8 to C16 and C17 (4.05 +/- 0.25 A) and from C8 to C18 (2.95 +/- 0.15 A), show that the major portion of retinylidene in rhodopsin has a twisted 6-s-cis conformation. The more precise distance measurement made here between C8 and C18 (2.95 A) predicts that the chain is twisted out-of-plane with respect to the ring by a modest amount (C5-C6-C7-C8 torsion angle = -28 +/- 7 degrees ).

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. This result suggests that in the bathorhodopsin-to-BSI transition the highly strained all-trans chromophore has relaxed in the C(10)-C(11)=C(12)-C(13) region, but is still distorted near C(14). The low frequency of the 11,12 A(u) HOOP mode in BSI compared with that of lumirhodopsin and metarhodopsin I indicates weaker coupling between the 11H and 12H wags due to residual distortion of the BSI chromophore near C(11)=C(12). The C=NH(+) stretching mode in BSI at 1653 cm(-1) exhibits a normal deuteriation induced downshift of 23 cm(-1), implying that there is no significant structural rearrangement of the Schiff base counterion region in the transition of bathorhodopsin to BSI. However, a dramatic Schiff base environment change occurs in the BSI-to-lumirhodopsin transition, because the 1638 cm(-1) C=NH(+) stretching mode in lumirhodopsin is unusually low and shifts only 7 cm(-1) in D(2)O, suggesting that it has essentially no H-bonding acceptor. With these data we can for the first time compare and discuss the room temperature resonance Raman vibrational structure of all the key intermediates in visual excitation.

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. The coupled modes of the hydrogen-out-of plane bending vibrations (HOOPs) of the N-H and C(15)-H of the protonated Schiff base are detected as a broad band centered at 911 cm(-1) for BR. A set of bands at 1073, 1064, and 1056 cm(-1) for L is shown to arise from the coupling of the HOOP with the overtones of interacting water O-H vibrations. Interaction with water was shown by the decreased intensity of the HOOPs of L in H(2)(18)O and by the influence of mutants that have been shown to perturb specific internal water molecules in BR. In contrast, the HOOP band of initial BR was not affected by these mutations. In D85N, the coupled HOOP of BR is depleted, while the coupled HOOPs of L are shifted. The results indicate that the Schiff base interacts with water in the L state but in a different manner than in the BR state. Moreover, the effects of mutations suggest that cytoplasmic water close to Thr46 (Wat46) either interacts stronger with the Schiff base in L or that it is important in stabilizing another water that does.