We describe a two-dimensional solid-state NMR technique to investigate membrane protein topology under magic-angle spinning conditions. The experiment detects the rate of (1)H spin diffusion from the mobile lipids to the rigid protein. While spin diffusion within the rigid protein is fast, magnetization transfer in the mobile lipids is an inefficient and slow process. Qualitative analysis of (1)H spin-diffusion build-up curves from the lipid chain-end methyl groups to the protein allows the identification of membrane-embedded domains in the protein. Numerical simulations of spin-diffusion build-up curves yield the approximate insertion depth of protein segments in the membrane. The experiment is demonstrated on the selectively (13)C labeled colicin Ia channel domain, known to have a membrane-embedded domain, and on DNA/cationic lipid complexes where the DNA rods are bound to the membrane surface. The experiment is designed for X-nucleus detection, which could be (13)C or (15)N in the protein and (31)P for the DNA. Finally, we show that a qualitative distinction between membrane proteins with and without a membrane-embedded domain can be made even by using an unlabeled protein, by detection of lipid signals. This spin-diffusion experiment is simple to perform and requires no oriented bilayer preparations and only standard NMR hardware.

We investigated lateral lipid organization in membranes with a lipid composition relevant to neural and retinal membranes [phosphatidylcholine (PC)/phosphatidylethanolamine (PE)/phosphatidylserine (PS)/cholesterol, 4/4/1/1, mol/mol/mol/mol]. The mixed-chain phospholipids contained saturated stearic acid (18:0) in the sn-1 position and the monounsaturated oleic acid (18:1) or polyunsaturated docosahexaenoic acid (22:6) in sn-2. Lateral lipid organization was evaluated by 2H NMR order parameter measurements on stearic acid of all individual types of phospholipids in the mixture and, through a novel approach, two-dimensional NOESY 1H NMR spectroscopy with magic angle spinning (MAS). The docosahexaenoic acid chain order was evaluated from 1H NMR chain signal MAS-sideband intensities. Averaged over all lipids, the cholesterol-induced increase in sn-1 chain order is 2-fold larger in monounsaturated than in polyunsaturated lipids, and the order of both saturated and polyunsaturated hydrocarbon chains increases. Addition of cholesterol increases lipid order in the sequence 18:0-18:1 PE > 18:0-18:1 PC > 18:0-18:1 PS for the monounsaturated and 18:0-22:6 PC >> 18:0-22:6 PE > 18:0-22:6 PS for polyunsaturated mixtures. The variation of order parameters between lipid species suggests that cholesterol induces the formation of lipid microdomains with a headgroup and chain unsaturation-dependent lipid composition. The preferential interaction between cholesterol and polyunsaturated 18:0-22:6 PC, followed by 18:0-22:6 PE and 18:0-22:6 PS, was confirmed by 1H MAS NOESY cross-relaxation rate differences. Furthermore, cholesterol preferentially associates with saturated chains in mixed-chain lipids reflected by higher saturated chain-to-cholesterol cross-relaxation rates. We propose that cholesterol forms PC-enriched microdomains in the polyunsaturated 18:0-22:6 PC/18:0-22:6 PE/18:0-22:6 PS/cholesterol membranes in which the saturated sn-1 chains are preferentially oriented toward the cholesterol molecules.

The cross-peaks between lipid resonances in two-dimensional nuclear Overhauser enhancement spectroscopy 1H NMR spectra, recorded with magic-angle spinning, contain valuable information about the structure and dynamics of the lipid bilayer. We have attempted a quantitative analysis of magnetization exchange between lipid resonances in the biologically relevant liquid crystalline lamellar phase. Spectra of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) water dispersions were recorded at mixing times from 0.005 to 1 s, and all diagonal and cross-peak volumes were determined by integration. The full relaxation-rate matrix was computed for the 10 lipid resonances by a matrix equation algorithm. Results of this mathematically rigorous approach were compared with simplified approaches to calculate cross-relaxation rates. In a second series of experiments, the DMPC was mixed with increasing amounts of perdeuterated DMPC-d 67 to determine the percentage of intra- vs intermolecular magnetization transfer. With the exception of transfer between nearest neighbor protons by chemical bond, cross-relaxation in DMPC bilayers is exclusively intermolecular. There is no evidence for the transfer of magnetization over several bonds within lipid molecules by spin diffusion. Lipids exist in multiple conformations with rapid transitions between them, and, therefore, cross-relaxation rates do not represent stable molecular arrangements with fixed distances. Instead, the cross-relaxation rates reflect the probability of close approach between protons of neighboring lipid molecules. We established experimentally that cross-relaxation rates are proportional to the statistical average of lateral lipid−lipid contacts in binary mixtures. The location of lipid segments along the bilayer normal is best described by a distribution function. Segments which are located at the same depth in the bilayer approach each other with higher probability and have higher rates of magnetization transfer, compared with groups which are, on average, more distant and approach each other less frequently. The per-proton intermolecular cross-relaxation rates vary over the bilayer by only 1 order of magnitude, indicating surprisingly high probabilities of close approach even between the most distant groups, like the choline methyl groups and the terminal methyl groups of hydrocarbon chains. The results reflect the high degree of lipid motional disorder and the substantial variations in location of neighboring lipid molecules.

Neuropeptide Y (NPY) receptors belong to the G protein-coupled receptor (GPCR) superfamily and play important roles in food intake, anxiety and cancer regulation1,2. The NPY/Y receptor system has emerged as one of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in mammals, namely Y1, Y2, Y4 and Y5 receptors, with different affinity and selectivity3. NPY is the most powerful stimulant of food intake and this effect is primarily mediated by Y1 receptor (Y1R)4. A number of peptides and small-molecule compounds have been characterized as Y1R antagonists and have shown clinical potential in the treatment of obesity4, tumor1 and bone loss5. However, their clinical usage has been hampered by low potency and selectivity, poor brain penetration ability or lack of oral bioavailability6. Here we report crystal structures of the human Y1R bound to two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 Å resolution, respectively. The structures combined with mutagenesis studies reveal binding modes of Y1R to several structurally diverse antagonists and determinants of ligand selectivity. The Y1R structure and molecular docking of the endogenous agonist NPY, together with nuclear magnetic resonance (NMR), photo-crosslinking and functional studies, provide insights into the binding behavior of the agonist and for the first time determine the interaction of its N terminus with the receptor. These insights into Y1R can enable structure-based drug discovery targeting NPY receptors.

Diffusion-controlled water permeation across bilayers of polyunsaturated phospholipids was measured by 17O nuclear magnetic resonance. In 100-nm extruded liposomes containing 50 mM MnCl2, water exchange between internal and external solutions was monitored via changes in the linewidth of the 17O water resonance of external water. Liposome size and shape were characterized by light scattering methods and determination of liposome trapped volume. At 25 degrees C, the following water permeability coefficients were determined: 18:0-18:1n-9 PC, 155 +/- 24 microns/s; 18:0-18:3n-3 PC, 330 +/- 88 microns/s; and 18:0-22:6n-3 PC, 412 +/- 91 microns/s. The addition of 1 M ethanol reduced permeability coefficients to 66 +/- 15 microns/s for 18:0-18:1n-9 PC and to 239 +/- 67 microns/s for 18:0-22:6n-3 PC. Furthermore, the addition of 50 mol% 18:1n-9-18:1n-9 PE reduced the water permeability from 122 +/- 21 microns/s for pure 18:1n-9-18:1n-9 PC to 74 +/- 15 microns/s for the mixture. The significant increase in water permeation for membranes with polyunsaturated hydrocarbon chains correlates with looser packing of polyunsaturated lipids at the lipid-water interface and the suggested deeper penetration of water into these bilayers. Ethanol may block water diffusion pathways by occupying points of water entry into bilayers at the interface. The addition of dioleoylphosphatidylethanolamine increases lipid packing density and, consequently, reduces permeation rates.

Cholesterol analogs are often used to investigate lipid trafficking and membrane organization of native cholesterol. Here, the potential of various spin (doxyl moiety) and fluorescent (7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group) labeled cholesterol analogs as well as of fluorescent cholestatrienol and the naturally occurring dehydroergosterol to mimic the unique properties of native cholesterol in lipid membranes was studied in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes by electron paramagnetic resonance, nuclear magnetic resonance, and fluorescence spectroscopy. As cholesterol, all analogs undergo fluctuating motions of large amplitude parallel to the bilayer normal. Native cholesterol keeps a strict orientation in the membrane with the long axis parallel to the bilayer normal. Depending on the chemical modification or the position of the label, cholesterol analogs may adopt an "up-sidedown" orientation in the membrane or may even fluctuate between "upright" and up-side-down orientation by rotational motions about the short axis not typical for native cholesterol. Those analogs are not able to induce a comparable condensation of phospholipid membranes as known for native cholesterol revealed by 2 H nuclear magnetic resonance. However, cholesterol-induced lipid condensation is one of the key properties of native cholesterol, and, therefore, a well suited parameter to assess the potential of steroid analogs to mimic cholesterol. The study points to extreme caution when studying cholesterol behavior by the respective analogs. Among seven analogs investigated, only a spin-labeled cholesterol with the doxyl group at the end of the acyl chain and the fluorophore cholestatrienol mimic cholesterol satisfactorily. Dehydroergosterol has a similar upright orientation as cholesterol and could be used at low concentration (about 1 mol %), at which its lower potential to enhance lipid packing density does not perturb membrane organization.Cholesterol constitutes a major component of mammalian cellular membranes. Its correct distribution among intracellular membranes and the plasma membrane is essential for the homeostasis of mammalian cells. Thus, intracellular trafficking plays a major role in the correct disposition of internalized cholesterol and in the regulation of cholesterol efflux (for a recent review, see Ref.

Solid-state NMR spectroscopy was employed to study the molecular dynamics of the colicin Ia channel domain in the soluble and membrane-bound states. In the soluble state, the protein executes small-amplitude librations (with root-mean-square angular fluctuations of 0-10 degrees ) in the backbone and larger-amplitude motions (16-17 degrees ) in the side chains. Upon membrane binding, the motional amplitudes increase significantly for both the backbone (12-16 degrees ) and side chains (23-29 degrees ), as manifested by the reduction in the C-H and H-H dipolar couplings and (15)N chemical shift anisotropy. These motions occur not only on the pico- to nanosecond time scales, but also on the microsecond time scale, as revealed by the (1)H rotating-frame spin-lattice relaxation times. Average motional correlation times of 0.8 and 1.2 micros were extracted for the soluble and membrane-bound states, respectively. In comparison, both forms of the colicin Ia channel domain are completely immobile on the millisecond scale. These results indicate that the colicin Ia channel domain has enhanced conformational mobility in the lipid bilayer compared to the soluble state. This membrane-induced mobility increase is consistent with the loss of tertiary structure of the protein in the membrane, which was previously suggested by the extended helical array model [Zakharov et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4282-4287]. An extended structure would also facilitate protein interactions with the mobile lipids and thus increase the protein internal motions. We speculate that the large mobility of the membrane-bound colicin Ia channel domain is a prerequisite for channel opening in the presence of a voltage gradient.

The interactions between glycosaminoglycans (GAGs), important components of the extracellular matrix, and proteins such as growth factors and chemokines play critical roles in cellular regulation processes. Therefore, the design of GAG derivatives for the development of innovative materials with bio-like properties in terms of their interaction with regulatory proteins is of great interest for tissue engineering and regenerative medicine. Previous work on the chemokine interleukin-8 (IL-8) has focused on its interaction with heparin and heparan sulfate, which regulate chemokine function. However, the extracellular matrix contains other GAGs, such as hyaluronic acid (HA), dermatan sulfate (DS) and chondroitin sulfate (CS), which have so far not been characterized in terms of their distinct molecular recognition properties towards IL-8 in relation to their length and sulfation patterns. NMR and molecular modeling have been in great part the methods of choice to study the structural and recognition properties of GAGs and their protein complexes. However, separately these methods have challenges to cope with the high degree of similarity and flexibility that GAGs exhibit. In this work, we combine fluorescence spectroscopy, NMR experiments, docking and molecular dynamics simulations to study the configurational and recognition properties of IL-8 towards a series of HA and CS derivatives and DS. We analyze the effects of GAG length and sulfation patterns in binding strength and specificity, and the influence of GAG binding on IL-8 dimer formation. Our results highlight the importance of combining experimental and theoretical approaches to obtain a better understanding of the molecular recognition properties of GAG–protein systems.