Selective interface detection: mapping binding site contacts in membrane proteins by NMR spectroscopy.

Intermolecular contact surfaces are important regions where specific interactions mediate biological function. We introduce a new magic angle spinning solid state NMR technique, dubbed "selective interface detection spectroscopy" (SIDY). In this technique, 13C-attached protons (1Hlig) are dephased by 1H-13C REDOR. A spin diffusion period is then used to enhance long distance 1H-1H correlations, and the results are detected by a short period of cross polarization to the 13C isotope labels. This SIDY approach allows selective observation of the interface between 13C-labeled and unlabeled moieties. We have used SIDY to probe the ligand-protein binding surface between a uniformly isotopically labeled ligand cofactor, U-13C20-11-cis-retinal, and its binding site in rhodopsin (Rho), an unlabeled, membrane-embedded G-protein coupled receptor (GPCR). The observed 1HGPCR-13Clig correlations indicate multiple close contacts between the protein and the ionone ring of the ligand, in agreement with binding studies. The polyene tail of the ligand displays fewer strong correlations in the SIDY spectrum. Some correlations can be assigned to the protein side chains lining the ligand binding site, in agreement with the crystal structure.

Accurate CSA measurements from uniformly isotopically labeled biomolecules at high magnetic field.

Obtaining chemical shift anisotropy (CSA) principal values from large biomolecular systems is often a laborious process of preparing many singly isotopically labeled samples and performing multiple independent CSA measurements. We present CSA tensor principal values measured in the biomolecular building blocks tyrosine.HCl, histidine.HCl, and all-E-retinal in both isotopically labeled and unlabeled forms at 17.6 T. The measured tensor values are identical for most carbon sites despite significant dipolar couplings between the spins. Quantum mechanical simulations of an arbitrary three spin system were used to evaluate the accuracy of direct CSA measurement as a function of applied magnetic field strength and molecular parameters. It was found that for a CSA asymmetry of 0.2 or more, an accurate measure of the CSA parameters is obtained when the CSA anisotropy is more than six times the largest dipolar coupling in frequency units. If the CSA asymmetry is more than 0.5, this requirement is relaxed, and accurate results are obtained if the anisotropy is more than three times the dipolar coupling. While these limits are insufficient for measurement of CSA's for alpha-carbons and aliphatic sidechain sites in proteins at current field strengths, they open the way for routine systematic CSA measurements of sites with relatively large CSA tensor values in extensively isotopically labeled biomolecules in widely available magnetic fields.

Solid-state NMR analysis of ligand--receptor interactions reveals an induced misfit in the binding site of isorhodopsin.

Rhodopsin is the photosensitive protein of the rod photoreceptor in the vertebrate retina and is a paradigm for the superfamily of G-protein-coupled receptors (GPCRs). Natural rhodopsin contains an 11-cis-retinylidene chromophore. We have prepared the 9-cis analogue isorhodopsin in a natural membrane environment using uniformly (13)C-enriched 9-cis retinal. Subsequently, we have determined the complete (1)H and (13)C assignments with ultra-high field solid-state magic angle spinning NMR. The 9-cis substrate conforms to the opsin binding pocket in isorhodopsin in a manner very similar to that of the 11-cis form in rhodopsin, but the NMR data reveal an improper fit of the 9-cis chromophore in this binding site. We introduce the term "induced misfit" to describe this event. Downfield proton NMR ligation shifts (Deltasigma(lig)(H) > 1 ppm) are observed for the 16,17,19-H and nearby protons of the ionone ring and for the 9-methyl protons. They provide converging evidence for global, nonspecific steric interactions between the chromophore and protein, and contrast with the specific interactions over the entire ionone ring and its substituents detected for rhodopsin. The Deltasigma(lig)(C) pattern of the polyene chain confirms the positive charge delocalization in the polyene associated with the protonation of the Schiff base nitrogen. In line with the misalignment of the ionone ring, an additional and anomalous perturbation of the (13)C response is detected in the region of the 9-cis bond. This provides evidence for strain in the isomerization region of the polyene and supports the hypothesis that perturbation of the conjugation around the cis bond induced by the protein environment assists the selective photoisomerization.

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.

(1)H and (13)C MAS NMR evidence for pronounced ligand-protein interactions involving the ionone ring of the retinylidene chromophore in rhodopsin.

Rhodopsin is a member of the superfamily of G-protein-coupled receptors. This seven alpha-helix transmembrane protein is the visual pigment of the vertebrate rod photoreceptor cells that mediate dim light vision. In the active binding site of this protein the ligand or chromophore, 11-cis-retinal, is covalently bound via a protonated Schiff base to lysine residue 296. Here we present the complete (1)H and (13)C assignments of the 11-cis-retinylidene chromophore in its ligand-binding site determined with ultra high field magic angle spinning NMR. Native bovine opsin was regenerated with 99% enriched uniformly (13)C-labeled 11-cis-retinal. From the labeled pigment, (13)C carbon chemical shifts could be obtained by using two-dimensional radio frequency-driven dipolar recoupling in a solid-state magic angle spinning homonuclear correlation experiment. The (1)H chemical shifts were assigned by two-dimensional heteronuclear ((1)H-(13)C) dipolar correlation spectroscopy with phase-modulated Lee-Goldburg homonuclear (1)H decoupling applied during the t(1) period. The data indicate nonbonding interactions between the protons of the methyl groups of the retinylidene ionone ring and the protein. These nonbonding interactions are attributed to nearby aromatic acid residues Phe-208, Phe-212, and Trp-265 that are in close contact with, respectively, H-16/H-17 and H-18. Furthermore, binding of the chromophore involves a chiral selection of the ring conformation, resulting in equatorial and axial positions for CH(3)-16 and CH(3)-17.

The preparation of all-trans uniformly (13)C-labeled retinal via a modular total organic synthetic strategy. emerging central contribution of organic synthesis toward the structure and function study with atomic resolution in protein research.

Uniformly [(13)C(20)]-labeled all-trans-retinal (1) has been prepared via a convergent modular total organic strategy with high isotope incorporation (>99%) and without isotope dilution starting from commercially available 99% enriched (13)C-labeled starting materials. For this purpose we have developed a strategy that is based on four different modules: [1,2,3,4,(3-CH(3))-(13)C(5)]-4-(diethylphosphono)-3-methyl-2-butenenitrile (3), [1,2,3,4-(13)C(4)]-ethyl acetoacetate (7), [U-(13)C(5)]-4-bromo-2-methyl-2-butene (13), and [U-(13)C(10)]-2,6,6-trimethylcyclohex-2-ene-1-ylcarbonitrile (16). This scheme permits the synthesis of the full cassette of all isotopomers with (13)C-labels at any position or combination of positions by using different (13)C-labeled starting materials. In addition, modifications of the synthesized modules will give access to a broad range of chemically modified (13)C-labeled retinoids and carotenoids. This modular strategy enables the synthesis of multifold and uniformly stable isotopically labeled (bio)macromolecules that can be used for studying proteins with atomic resolution, providing detailed functional information of the studied biological system.