The structure of the M2 channel-lining segment from the nicotinic acetylcholine receptor

The structures of functional peptides corresponding to the predicted channel-lining M2 segment of the nicotinic acetylcholine (AChR) were determined using solution NMR experiments on micelle samples, and solid-state NMR experiments on bilayer samples. The AChR M2 peptide forms a straight transmembrane alpha-helix, with no kinks. M2 inserts in the lipid bilayer at an angle of 12 degrees relative to the bilayer normal, with a rotation about the helix long axis such that the polar residues face the N-terminus of the peptide, which is assigned to be intracellular. A molecular model of the AChR channel pore, constructed from the solid-state NMR 3-D structure of the AChR M2 helix in the membrane assuming a pentameric organization, results in a funnel-like architecture for the channel with the wide opening on the N-terminal intracellular side. A central narrow pore has a diameter ranging from about 3.0 A at its narrowest, to 8.6 A at its widest. Nonpolar residues are predominantly on the exterior of the bundle, while polar residues line the pore. This arrangement is in fair agreement with evidence collected from permeation, mutagenesis, affinity labeling and cysteine accessibility measurements. A pentameric M2 helical bundle may, therefore, represent the structural blueprint for the inner bundle that lines the channel of the nicotinic AChR.     

The structure of the M2 channel-lining segment from the nicotinic acetylcholine receptor

The structures of functional peptides corresponding to the predicted channel-lining M2 segment of the nicotinic acetylcholine (AChR) were determined using solution NMR experiments on micelle samples, and solid-state NMR experiments on bilayer samples. The AChR M2 peptide forms a straight transmembrane α-helix, with no kinks. M2 inserts in the lipid bilayer at an angle of 12° relative to the bilayer normal, with a rotation about the helix long axis such that the polar residues face the N-terminus of the peptide, which is assigned to be intracellular. A molecular model of the AChR channel pore, constructed from the solid-state NMR 3-D structure of the AChR M2 helix in the membrane assuming a pentameric organization, results in a funnel-like architecture for the channel with the wide opening on the N-terminal intracellular side. A central narrow pore has a diameter ranging from about 3.0 Å at its narrowest, to 8.6 Å at its widest. Nonpolar residues are predominantly on the exterior of the bundle, while polar residues line the pore. This arrangement is in fair agreement with evidence collected from permeation, mutagenesis, affinity labeling and cysteine accessibility measurements. A pentameric M2 helical bundle may, therefore, represent the structural blueprint for the inner bundle that lines the channel of the nicotinic AChR.     

A structural and dynamic model for the nicotinic acetylcholine receptor

Folding of the five polypeptide subunits (alpha 2 beta gamma delta) of the nicotinic acetylcholine receptor (AChR) into a functional structural model is described. The principles used to arrange the sequences into a structure include: (1) hydrophobicity----membrane-crossing segments; (2) amphipathic character----ion-carrying segments (ion channel with single group rotations); (3) molecular shape (elongated, pentagonal cylinder)----folding dimensions of exobilayer portion; (4) choice of acetylcholine binding sites----specific folding of exobilayer segments; (5) location of reducible disulfides (near agonist binding site)----additional specification of exobilayer arrangement; (6) genetic homology----consistency of functional group choices; (7) noncompetitive antagonist labeling----arrangement of bilayer helices. The AChR model is divided into three parts: (a) exobilayer consisting of 11 antiparallel beta-strands from each subunit; (b) bilayer consisting of four hydrophobic and one amphiphilic alpha-helix from each subunit; (c) cytoplasmic consisting of one (folded) loop from each subunit. The exobilayer strands can form a closed 'flower' (the 'resting state') which is opened ('activated') by agonists bound perpendicular to the strands. Rearrangement of the agonists to a strand-parallel position and partial closing of the 'flower' leads to a desensitized receptor. The actions of acetylcholine and succinoyl and suberoyl bis-cholines are clarified by the model. The opening and closing of the exobilayer 'flower' controls access to the ion channel which is composed of the five amphiphilic bilayer helices. A molecular mechanism for ion flow in the channel is given. Openings interrupted by short duration closings (50 microseconds) depend upon channel group motions. The unusual photolabeling of intrabilayer serines in alpha, beta and delta subunits but not in gamma subunits near the binding site for non-competitive antagonists is explained along with a mechanism for the action of these antagonists such as phencyclidine. The unusual alpha 192Cys-193Cys disulfide may have a special peptide arrangement, such as a cis-peptide bond to a following proline (G.A. Petsko and E.M. Kosower, unpublished results). The position of phosphorylatable sites and proline-rich segments are noted for the cytoplasmic loops. The dynamic behavior of the AChR channel and many different experimental results can be interpreted in terms of the model. An example is the lowering of ionic conductivity on substitution of bovine for Torpedo delta M2 segment. The model represents a useful construct for the design of experiments on AChR.     

Chimeric muscarinic cholinergic: beta-adrenergic receptors that activate Gs in response to muscarinic agonists

The M1-muscarinic cholinergic receptor (M1AChR) stimulates the release of inositol phosphates (IPs) but does not activate adenylyl cyclase. The beta-adrenergic receptor (beta-AR) stimulates adenylyl cyclase but has no effect on IP release. Amino acid sequences corresponding to the second (I2) and third (I3) intracellular loops of the turkey erythrocyte beta-AR and a 12-amino acid segment near the N-terminal end of the I3 region were substituted into the corresponding regions of the human M1AChR. Chimeric receptors that contained either the entire I3 loop or the N-terminal dodecapeptide of that loop both mediated the 2-4-fold stimulation of adenylyl cyclase activity in membrane fractions of COS, A293, or Sf9 cells in response to carbachol. These chimeric receptors also retained the ability to stimulate IP release to the same extent as did the M1AChR. In COS cells transfected with the I3 chimeric receptor, the EC50 for carbachol was approximately 7 microM for the stimulation of adenylyl cyclase and approximately 2 microM for the release of IP; M1AChR-mediated IP release displayed an EC50 of approximately 0.2 microM. Substitution of the I2 region of the beta-AR into the M1AChR did not by itself alter selectivity for signaling. However, the I2+I3 and I2+dodecapeptide combined replacements stimulated adenylyl cyclase fully and caused at most 25% of the maximal stimulation of IP release observed with the M1AChR. Thus, a small region in the third cytoplasmic loop can alter the G proteins to which a receptor is coupled, but interaction among loops is evidently involved in fully determining G protein selectivity.     

Detection of G protein-activator regions in M4 subtype muscarinic, cholinergic, and alpha 2-adrenergic receptors based upon characteristics in primary structure.

The 14-residue region Arg2410-Lys2423 of the human insulin-like growth factor II receptor possesses the ability to stimulate Gi, the activity being dependent on two structural characteristics: (i) at least two basic residues at the N-terminal side and (ii) the C-terminal motif, B-B-X-B or B-B-X-X-B (where B is a basic residue and X is a non-basic residue). The regions satisfying (i) and (ii) with 10 less than or equal to residue length less than or equal to 26 were located in all of the third inner loops and some of the other intracellular domains of the Gi-coupled M4 sub-type muscarinic cholinergic receptor (M4AChR) and the alpha 2-adrenergic receptor (alpha 2AR). Both the second inner loop 130-147 and the C-terminal portion of the third inner loop 382-400 (MIII) of human M4AChR had the ability to stimulate G proteins with the order Gi approximately Go greater than Gs, but only MIII could activate Gi/Go at nanomolar concentrations. In contrast, the N-terminal portion of the third inner loop 218-228 of human alpha 2AR-C10 activated Gi, Go, and Gs at micromolar concentrations with equal potency, whereas the further C-terminal portion of the third inner loop 301-313 of this receptor lacked the ability to activate any G protein. Among these active regions, only MIII indicated Mg(2+)-dependent Gi-stimulating function. Therefore, the search for the regions satisfying (i) and (ii) was useful to localize the G protein-activating activity of Gi-coupled receptors in limited regions, which were not always in the C-terminal portions of the third intracellular loops and activated G proteins in various modes of actions.     

Nicotinic receptor fourth transmembrane domain: hydrogen bonding by conserved threonine contributes to channel gating kinetics

The fourth transmembrane domain (M4) of the nicotinic acetylcholine receptor (AChR) contributes to the kinetics of activation, yet its close association with the lipid bilayer makes it the outermost of the transmembrane domains. To investigate mechanistic and structural contributions of M4 to AChR activation, we systematically mutated alphaT422, a conserved residue that has been labeled by hydrophobic probes, and evaluated changes in rate constants underlying ACh binding and channel gating steps. Aromatic and nonpolar mutations of alphaT422 selectively affect the channel gating step, slowing the rate of opening two- to sevenfold, and speeding the rate of closing four- to ninefold. Additionally, kinetic modeling shows a second doubly liganded open state for aromatic and nonpolar mutations. In contrast, serine and asparagine mutations of alphaT422 largely preserve the kinetics of the wild-type AChR. Thus, rapid and efficient gating of the AChR channel depends on a hydrogen bond involving the side chain at position 422 of the M4 transmembrane domain.     

Structure,topology, and dynamics of myristoylated recoverin bound to phospholipid bilayers

Recoverin, a member of the EF-hand protein superfamily, serves as a calcium sensor in retinal rod cells. A myristoyl group covalently attached to the N-terminus of recoverin facilitates its binding to retinal disk membranes by a mechanism known as the Ca(2+)-myristoyl switch. Samples of (15)N-labeled Ca(2+)-bound myristoylated recoverin bind anisotropically to phospholipid membranes as judged by analysis of (15)N and (31)P chemical shifts observed in solid-state NMR spectra. On the basis of a (2)H NMR order parameter analysis performed on recoverin containing a fully deuterated myristoyl group, the N-terminal myristoyl group appears to be located within the lipid bilayer. Two-dimensional solid-state NMR ((1)H-(15)N PISEMA) spectra of uniformly and selectively (15)N-labeled recoverin show that the Ca(2+)-bound protein is positioned on the membrane surface such that its long molecular axis is oriented approximately 45 degrees with respect to the membrane normal. The N-terminal region of recoverin points toward the membrane surface, with close contacts formed by basic residues K5, K11, K22, K37, R43, and K84. This orientation of the membrane-bound protein allows an exposed hydrophobic crevice, near the membrane surface, to serve as a potential binding site for the target protein, rhodopsin kinase. Close agreement between experimental and calculated solid-state NMR spectra of recoverin suggests that membrane-bound recoverin retains the same overall three-dimensional structure that it has in solution. These results demonstrate that membrane binding by recoverin is achieved primarily by insertion of the myristoyl group inside the bilayer with apparently little rearrangement of the protein structure.     

Proline kink angle distributions for GWALP23 in lipid bilayers of different thicknesses

By using selected (2)H and (15)N labels, we have examined the influence of a central proline residue on the properties of a defined peptide that spans lipid bilayer membranes by solid-state nuclear magnetic resonance (NMR) spectroscopy. For this purpose, GWALP23 (acetyl-GGALW(5)LALALALALALALW(19)LAGA-ethanolamide) is a suitable model peptide that employs, for the purpose of interfacial anchoring, only one tryptophan residue on either end of a central α-helical core sequence. Because of its systematic behavior in lipid bilayer membranes of differing thicknesses [Vostrikov, V. V., et al. (2010) J. Biol. Chem. 285, 31723-31730], we utilize GWALP23 as a well-characterized framework for introducing guest residues within a transmembrane sequence; for example, a central proline yields acetyl-GGALW(5)LALALAP(12)ALALALW(19)LAGA-ethanolamide. We synthesized GWALP23-P12 with specifically placed (2)H and (15)N labels for solid-state NMR spectroscopy and examined the peptide orientation and segmental tilt in oriented DMPC lipid bilayer membranes using combined (2)H GALA and (15)N-(1)H high-resolution separated local field methods. In DMPC bilayer membranes, the peptide segments N-terminal and C-terminal to the proline are both tilted substantially with respect to the bilayer normal, by ~34 ± 5° and 29 ± 5°, respectively. While the tilt increases for both segments when proline is present, the range and extent of the individual segment motions are comparable to or smaller than those of the entire GWALP23 peptide in bilayer membranes. In DMPC, the proline induces a kink of ~30 ± 5°, with an apparent helix unwinding or "swivel" angle of ~70°. In DLPC and DOPC, on the basis of (2)H NMR data only, the kink angle and swivel angle probability distributions overlap those of DMPC, yet the most probable kink angle appears to be somewhat smaller than in DMPC. As has been described for GWALP23 itself, the C-terminal helix ends before Ala(21) in the phospholipids DMPC and DLPC yet remains intact through Ala(21) in DOPC. The dynamics of bilayer-incorporated, membrane-spanning GWALP23 and GWALP23-P12 are less extensive than those observed for WALP family peptides that have more than two interfacial Trp residues.     

Structure of monomeric Interleukin-8 and its interactions with the N-terminal Binding Site-I of CXCR1 by solution NMR spectroscopy

The structure of monomeric human chemokine IL-8 (residues 1–66) was determined in aqueous solution by NMR spectroscopy. The structure of the monomer is similar to that of each subunit in the dimeric full-length protein (residues 1–72), with the main differences being the location of the N-loop (residues 10–22) relative to the C-terminal α-helix and the position of the side chain of phenylalanine 65 near the truncated dimerization interface (residues 67–72). NMR was used to analyze the interactions of monomeric IL-8 (1–66) with ND-CXCR1 (residues 1–38), a soluble polypeptide corresponding to the N-terminal portion of the ligand binding site (Binding Site-I) of the chemokine receptor CXCR1 in aqueous solution, and with 1TM-CXCR1 (residues 1–72), a membrane-associated polypeptide that includes the same N-terminal portion of the binding site, the first trans-membrane helix, and the first intracellular loop of the receptor in nanodiscs. The presence of neither the first transmembrane helix of the receptor nor the lipid bilayer significantly affected the interactions of IL-8 with Binding Site-I of CXCR1.     

Conformation of cytoplasmic segments of acetylcholine receptor alpha- and beta-subunits probed by monoclonal antibodies: sensitivity of the antibody competition approach.

The conformation of the cytoplasmic side of Torpedo marmorata acetylcholine receptor (AChR) was investigated by 22 monoclonal antibodies (mAbs) binding to known sites on the amino acid sequences 339-378 and 336-469 of the AChR alpha- and beta-subunits respectively. Competitions among these mAbs for binding on the intact AChR were compared with their competition for binding on the SDS-denatured subunits and with their corresponding epitopes previously determined on the primary structure of the subunits. We found the following: The three approaches correlated very well suggesting that these mAbs bind on the intact AChR at the same sequences determined by synthetic peptides and not on irrelevant discontinuous epitopes; this finding supports conclusions of Ratnam et al. (1986a) that the amphipathic helix M5 is exposed on the cytoplasmic side of the AChR. The subunit segments alpha 339-378 and beta 336-469 seem to be extended over large distances on the cytoplasmic surface of the AChR. The cytoplasmic surface of beta-subunit has a very immunogenic region. The mAb-competition technique is very sensitive since mAbs to epitopes separated by only about seven residues did not exclude each other, and mAbs to overlapping epitopes exhibited differential competitions with other mAbs.     

The membrane interactions of antimicrobial peptides revealed by solid-state NMR spectroscopy

Solid-state NMR spectroscopic techniques provide valuable information about the structure, dynamics and topology of membrane-inserted polypeptides. In particular antimicrobial peptides (or 'host defence peptides') have early on been investigated by solid-state NMR spectroscopy and many technical innovations in this domain have been developed with the help of these compounds when reconstituted into oriented phospholipid bilayers. Using solid-state NMR spectroscopy it could be shown for the first time that magainins or derivatives thereof exhibit potent antimicrobial activities when their cationic amphipathic helix is oriented parallel to the bilayer surface, a configuration found in later years for many other linear cationic amphipathic peptides. In contrast transmembrane alignments or lipid-dependent tilt angles have been found for more hydrophobic sequences such as alamethicin or β-hairpin antimicrobials. This review presents various solid-state NMR approaches and develops the basic underlying concept how angular information can be obtained from oriented samples. It is demonstrated how this information is used to calculate structures and topologies of peptides in their native liquid-disordered phospholipid bilayer environment. Special emphasis is given to discuss which NMR parameters provide the most complementary information, the minimal number of restraints needed and the effect of motions on the analysis of the NMR spectra. Furthermore, recent (31)P and (2)H solid-state NMR measurements of lipids are presented including some unpublished data which aim at investigating the morphological and structural changes of oriented or non-oriented phospholipids. Finally the structural models that have been proposed for the mechanisms of action of these peptides will be presented and discussed in view of the solid-state NMR and other biophysical experiments.     

Structural and dynamic studies of the gamma-M4 trans-membrane domain of the nicotinic acetylcholine receptor

A structural characterization of a synthetic peptide corresponding to the fourth transmembrane domain (M4-TMD) of the gamma-subunit of the nicotinic acetylcholine receptor from Torpedo californica has been undertaken. Solid-state NMR and CD spectroscopy studies indicate that upon reconstitution into lipid vesicles or magnetically aligned lipid bilayers, the synthetic M4-TMD adopts a linear alpha-helical conformation with the helix aligned within 15 degrees of the membrane normal. Furthermore, analysis of the motional averaging of anisotropic interactions present in the solid-state NMR spectra of the reconstituted peptide, indicate that the dynamics of the peptide within the bilayer are highly sensitive to the phase adopted by the lipid bilayer, providing an insight into how the interaction of lipids with this domain may play a important role in the modulation of this receptor by its lipid environment.     

Dynamics of fd coat protein in the bacteriophage

The dynamics of the coat protein in fd bacteriophage are described with solid-state 15N and 2H NMR experiments. The virus particles and the coat protein subunits are immobile on the time scales of the 15N chemical shift anisotropy (10(3) Hz) and 2H quadrupole (10(6) Hz) interactions. Previously we have shown that the Trp-26 side chain is immobile, that the two Tyr and three Phe side chains undergo only rapid twofold jump motions about their C beta-C gamma bond axis [Gall, C. M., Cross, T. A., DiVerdi, J. A., & Opella, S. J. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 101-105], and that most of the backbone peptide linkages are highly constrained but do undergo rapid small amplitude motions [Cross, T. A., & Opella, S. J. (1982) J. Mol. Biol. 159, 543-549] in the coat protein subunits in the virus particles. In this paper, we demonstrate that the four N-terminal residues of the coat protein subunits are highly mobile, since both backbone and side-chain sites of these residues undergo large amplitude motions that are rapid on the time scales of the solid-state NMR experiments. In addition, the dynamics of the methyl-containing aliphatic residues Ala, Leu, Val, Thr, and Met are analyzed. Large amplitude jump motions are observed in nearly all of these side chains even though, with the exception of the N-terminal residue Ala-1, their backbone peptide linkages are highly constrained. The established information about the dynamics of the structural form of fd coat protein in the virus particle is summarized qualitatively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid matters: nicotinic acetylcholine receptor-lipid interactions (Review)

Ligand-gated ion channels mediate fast intercellular communication in response to endogenous neurotransmitters. The nicotinic acetylcholine receptor (AChR) is the archetype molecule in the superfamily of these membrane proteins. Early electron spin resonance studies led to the discovery of a lipid fraction in direct contact with the AChR, with rotational dynamics 50-fold slower than those of the bulk lipids. This AChR-vicinal lipid region has since been postulated to be a possible site of lipid modulation of receptor function. The polarity and molecular dynamics of solvent dipoles - mainly water - of AChR-vicinal lipids in the membrane have been studied with Laurdan extrinsic fluorescence, and Förster-type resonance energy transfer (FRET) was introduced to characterize the receptor-associated lipid microenvironment. FRET enabled one to discriminate between the bulk lipid and the AChR-vicinal lipid. The latter is in a liquid-ordered phase and exhibits a higher degree of order than the bulk bilayer lipid. Changes in FRET efficiency induced by fatty acids, phospholipids and cholesterol also led to the identification of discrete sites for these lipids on the AChR protein. After delineating the topography of the AChR membrane-embedded domains with fluorescence methods, sites for steroids are being explored with site-directed mutagenesis and patch-clamp recording. Pyrene-labelled Cys residues in &#102 M1, &#102 M4, &#110 M1 and &#110 M4 transmembrane regions were found to lie in a shallow position. For M4 segments, this is in agreement with a canonical linear &#102 -helix; for M1, it is necessary to postulate a substantial amount of non-helical structure, and/or of kinks, to rationalize the shallow location of Cys residues. Mutations of Thr 422, a residue close to the extracellular-facing membrane hemilayer in &#102 M4, affect the steroid modulation of AChR function, suggesting its involvement in steroid-AChR interactions.     

Insights on the interactions of synthetic amphipathic peptides with model membranes as revealed by 31P and 2H solid-state NMR and infrared spectroscopies

We studied the interaction between synthetic amphipathic peptides and model membranes by solid-state NMR and infrared spectroscopies. Peptides with 14 and 21 amino acids composed of leucines and phenylalanines modified by the addition of crown ethers were synthesized. The 14-mer and 21-mer peptides both possess a helical amphipathic structure. To shed light on their membrane interaction, (31)P and (2)H solid-state NMR experiments were performed on both peptides in interaction with dimyristoylphosphatidylcholine vesicles in the absence and presence of cholesterol, dimyristoylphosphatidylglycerol vesicles, and oriented bicelles. (31)P NMR experiments on multilamellar vesicles reveal that the dynamics and/or orientation of the polar headgroups are weakly yet markedly affected by the presence of the peptides, whereas (31)P NMR experiments on bicelles indicate no significant changes in the morphology and orientation of the bicelles. On the other hand, (2)H NMR experiments on vesicles reveal that the acyl chain order is affected differently depending on the membrane lipidic composition and on the peptide hydrophobic length. Finally, infrared spectroscopy was used to study the interfacial region of the bilayer. Based on these studies, mechanisms of membrane perturbation are proposed for the 14-mer and 21-mer peptides in interaction with model membranes depending on the bilayer composition and peptide length.     

An innovative procedure using a sublimable solid to align lipid bilayers for solid-state NMR studies

Uniaxially aligned phospholipid bilayers are often used as model membranes to obtain structural details of membrane-associated molecules, such as peptides, proteins, drugs, and cholesterol. Well-aligned bilayer samples can be difficult to prepare and no universal procedure has been reported that orients all combinations of membrane-embedded components. In this study, a new method for producing mechanically aligned phospholipid bilayer samples using naphthalene, a sublimable solid, was developed. Using (31)P-NMR spectroscopy, comparison of a conventional method of preparing mechanically aligned samples with the new naphthalene procedure found that the use of naphthalene significantly enhanced the alignment of 3:1 1-palmitoyl-2-oleoyl-phosphatidylethanolamine to 1-palmitoyl-2-oleoyl-phosphatidylglycerol. The utility of the naphthalene procedure is also demonstrated on bilayers of many different compositions, including bilayers containing peptides such as pardaxin and gramicidin. These results show that the naphthalene procedure is a generally applicable method for producing mechanically aligned samples for use in NMR spectroscopy. The increase in bilayer alignment implies that this procedure will improve the sensitivity of solid-state NMR experiments, in particular those techniques that detect low-sensitivity nuclei, such as 15N and 13C.     

Interactions of interleukin-8 with the human chemokine receptor CXCR1 in phospholipid bilayers by NMR spectroscopy

CXCR1 is a receptor for the chemokine interleukin-8 (IL-8), a mediator of immune and inflammatory responses. Strategically located in the cell membrane, CXCR1 binds to IL-8 with high affinity and subsequently transduces a signal across the membrane bilayer to a G-protein-activated second messenger system. Here, we describe NMR studies of the interactions between IL-8 and human CXCR1 in lipid environments. Functional full-length and truncated constructs of CXCR1 and full-length IL-8 were uniformly ¹⁵N-labeled by expression in bacteria followed by purification and refolding. The residues responsible for interactions between IL-8 and the N-terminal domain of CXCR1 were identified by specific chemical shift perturbations of assigned resonances on both IL-8 and CXCR1. Solution NMR signals from IL-8 in q = 0.1 isotropic bicelles disappeared completely when CXCR1 in lipid bilayers was added in a 1:1 molar ratio, indicating that binding to the receptor-containing bilayers immobilizes IL-8 (on the ∼ 10⁵ Hz timescale) and broadens the signals beyond detection. The same solution NMR signals from IL-8 were less affected by the addition of N-terminal truncated CXCR1 in lipid bilayers, demonstrating that the N-terminal domain of CXCR1 is mainly responsible for binding to IL-8. The interaction is tight enough to immobilize IL-8 along with the receptor in phospholipid bilayers and is specific enough to result in well-aligned samples in oriented sample solid-state NMR spectra. A combination of solution NMR and solid-state NMR studies of IL-8 in the presence of various constructs of CXCR1 enables us to propose a model for the multistep binding process.     

Solid-state NMR studies of the membrane-bound closed state of the colicin E1 channel domain in lipid bilayers.

The colicin E1 channel polypeptide was shown to be organized anisotropically in membranes by solid-state NMR analysis of samples of uniformly 15N-labeled protein in oriented planar phospholipid bilayers. The 190 residue C-terminal colicin E1 channel domain is the largest polypeptide to have been characterized by 15N solid-state NMR spectroscopy in oriented membrane bilayers. The 15N-NMR spectra of the colicin E1 show that: (1) the structure and dynamics are independent of anionic lipid content in both oriented and unoriented samples; (2) assuming the secondary structure of the polypeptide is helical, there are both trans-membrane and in-plane helical segments; (3) trans-membrane helices account for approximately 20-25% of the channel polypeptide, which is equivalent to 38-48 residues of the 190-residue polypeptide. The results of the two-dimensional PISEMA spectrum are interpreted in terms of a single trans-membrane helical hairpin inserted into the bilayer from each channel molecule. These data are also consistent with this helical hairpin being derived from the 38-residue hydrophobic segment near the C-terminus of the colicin E1 channel polypeptide.     

Macromolecular structural elucidation with solid-state NMR-derived orientational constraints

Summary The complete structure determination of a polypeptide in a lipid bilayer environment is demonstrated built solely upon orientational constraints derived from solid-state NMR observations. Such constraints are obtained from isotopically labeled samples uniformly aligned with respect to the B₀ field. Each observation constrains the molecular frame with respect to B₀ and the bilayer normal, which are arranged to be parallel. These constraints are not only very precise (a few tenths of a degree), but also very accurate. This is clearly demonstrated as the back bone structure is assembled sequentially and the i to i+6 hydrogen bonds in this structure of the gramicidin channel are shown on average to be within 0.5 Å of ideal geometry. Similarly, the side chains are assembled independently and in a radial direction from the backbone. The lack of considerable atomic overlap between side chains also demonstrates the accuracy of the constraints. Through this complete structure, solid-state NMR is demonstrated as an approach for determining three-dimensional macromolecular structure.     

Lipid matters: nicotinic acetylcholine receptor-lipid interactions (Review)

Ligand-gated ion channels mediate fast intercellular communication in response to endogenous neurotransmitters. The nicotinic acetylcholine receptor (AChR) is the archetype molecule in the superfamily of these membrane proteins. Early electron spin resonance studies led to the discovery of a lipid fraction in direct contact with the AChR, with rotational dynamics 50-fold slower than those of the bulk lipids. This AChR-vicinal lipid region has since been postulated to be a possible site of lipid modulation of receptor function. The polarity and molecular dynamics of solvent dipoles-mainly water-of AChR-vicinal lipids in the membrane have been studied with Laurdan extrinsic fluorescence, and Forster-type resonance energy transfer (FRET) was introduced to characterize the receptor-associated lipid microenvironment. FRET enabled one to discriminate between the bulk lipid and the AChR-vicinal lipid. The latter is in a liquid-ordered phase and exhibits a higher degree of order than the bulk bilayer lipid. Changes in FRET efficiency induced by fatty acids, phospholipids and cholesterol also led to the identification of discrete sites for these lipids on the AChR protein. After delineating the topography of the AChR membrane-embedded domains with fluorescence methods, sites for steroids are being explored with site-directed mutagenesis and patch-clamp recording. Pyrene-labelled Cys residues in alphaM1, alphaM4, gammaM1 and gammaM4 transmembrane regions were found to lie in a shallow position. For M4 segments, this is in agreement with a canonical linear alpha-helix; for M1, it is necessary to postulate a substantial amount of non-helical structure, and/or of kinks, to rationalize the shallow location of Cys residues. Mutations of Thr422, a residue close to the extracellular-facing membrane hemilayer in alphaM4, affect the steroid modulation of AChR function, suggesting its involvement in steroid-AChR interactions.