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Membranes Studied by NMR Spectroscopy*
Membranes Studied by NMR Spectroscopy A Watts, University of Oxford, UK SJ Opella, University of Pennsylvania, Philadelphia, PA, USA & 1999 Elsevier Ltd. All rights reserved. This article is reproduced from the previous edition, volume 2, pp 1281–1291, & 1999, Elsevier Ltd.
Introduction Molecular motions in biomembranes are highly anisotropic. Since essentially all aspects of NMR spectroscopy are affected by molecular motions, the details of these motions strongly influence the spectroscopic approaches that can be applied to membrane samples. Although the motions of individual molecules or molecular groups may be very rapid (the molecular correlation time, tc, is in the ns range for lipid hydrocarbon chains and amino acid side chains of proteins), the overall tumbling of the lipid and polypeptide molecules in membrane bilayers is very slow and can be in the ms or s range. For this reason, conventional solution-state NMR, which relies on rapid overall molecular reorientation to narrow the resonance lines, has found little success in membrane research; even a lipid molecule with a relative molecular mass (Mr) of B1000 or a small peptide (MrB3000) has, when in membrane bilayers, by solution-state NMR criteria a tumbling rate equivalent to a very large (Mrc30 kDa) macromolecule in free solution. Only by sonicating the sample to produce small (diameter 20–50 nm) bilayer vesicles can conventional solution-state NMR methods be used. However, not only are there very serious concerns about the status of the lipids and polypeptides embedded in highly distorted lipid bilayers, but also those portions of proteins embedded in the bilayers may still reorient too slowly to yield resolvable resonances. Solution-state NMR spectroscopy has been applied to peptides and proteins in organic solvents, detergent micelles and bicelles (Figure 1), and structures have been determined in these model membrane environments. While micelles certainly provide a more relevant environment for membrane proteins than do mixtures of organic solvents, fully hydrated lipid bilayers remain the definitive environment for structural and functional studies of membrane proteins. As an alternative approach to solution-state NMR methods, which are ineffective with lipid bilayer samples, solid-state NMR methods have been refined sufficiently to permit structural details to be obtained for membraneembedded peptides and proteins. This usually requires isotopic enrichment, either through chemical synthesis or through biosynthetic incorporation in expressed peptides and proteins. In the absence of routine X-ray
Figure 1 Representation of the types of sample preparations used for studying membrane lipids and proteins. Micelles and bicelles are usually small (diameters B20–50 nm) structures and bilayers can be sonicated into small (20–50 nm diameter) vesicles, or produced as extended (diametersc100 nm) multibilayered or single bilayered closed or open structures, depending upon the method of preparation. Natural membranes are usually as large bilayer fragments or closed structures containing a complex and heterogeneous mixture of lipids and proteins and possibly carbohydrates.
crystallographic structural studies for these molecules, solid-state NMR spectroscopy has the potential to be a powerful and unique approach to determining the structures and describing the dynamics and functions of membranes and membrane-bound proteins. In addition, solid-state NMR spectroscopy has been widely used to describe lipid structure, dynamics and phase properties. Thus, solid-state NMR experiments can be applied to both the polypeptide and lipid components of membrane bilayers, without the need to disrupt the sample through sonication or the addition of organic solvents or detergents.
Nuclei Used in Membrane Studies With the exception of J-couplings, the major magnetic interactions (chemical shift, dipolar and quadrupolar couplings) for the nuclei exploited in biological NMR can be averaged with respect to the applied fields (B0 BMHz and B1BkHz) by isotropic molecular motion of small molecules (Table 1). However, for biomembranes, any of these interactions may yield resonances with very broad lines and dominate the spectra, masking the resolution required for high resolution studies. Where these
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interactions can be exploited, their anisotropy (usually chemical shift, dipolar or quadrupolar) can give molecular orientational information from static samples, either oriented or as random dispersions (see below). Alternatively, magic angle spinning (MAS) of the sample can be used at spinning speeds (or) which are either fast enough to average the interaction completely (orcs, D, Q) to give high resolution-like solid-state NMR spectra, or may be moderated either to recouple a dipolar interaction, such as in rotational resonance or REDOR, or to provide orientionally dependent spinning spectral side-bands for nuclei which display chemical shift anisotropy (e.g. 31P, 15N). Naturally occurring 13C (natural abundance and with selective enrichment) and 31P nuclei have been extensively exploited in membrane NMR studies (Table 2). However, replacement of 1H by 2H or 19F, and 14N by 15 N, has also found widespread application, although to date 17O has not found application in these systems. Typical spectra for the more commonly exploited nuclei for lipids in bilayers are shown in Figure 2. The need to average the strong dipolar coupling (B100 kHz) for 1H to obtain high resolution spectra has excluded widespread observation of this nucleus in membranes. Extensive protein deuteration, to leave a minor 1H density at a site of interest for observation in micellar suspensions, has been achieved. The realization that reorientation around the long molecular axis rotation of lipids and proteins in membrane bilayers in the liquid crystalline phase is sufficiently fast (at about 109 Hz for lipids and 106 Hz for proteins with radius r4 nm in fluid membranes) to average even homonuclear 1H dipolar couplings, has opened a new avenue for membrane studies for most observable nuclei, including 1H without the need for isotopic replacements. In addition, it is possible to perform magic angle oriented sample spinning (MAOSS) experiments to reap the benefits of both sample orientation and magic angle sample spinning in this situation.
Table 1 Comparison of the strengths of the magnetic interactions in NMR and the ways in which they can be averaged to yield molecular information for lipids, peptides and proteins in membranes in liquid state and solid state approaches Interaction
Liquids (Hz)
Solids (Hz)
Methods
s J D Q
10–104 102 0 0
10–104 102 104 105–106
MAS Decoupling Decoupling, MAS MAS
Adapted with permission from Smith SO, Ascheim K, and Groesbeck M (1996) Magic angle spinning NMR spectroscopy of membrane proteins. Quarterly Review of Biophysics 29: 395–449. s, Chemical shift anisotropy; J, J-coupling; D, dipolar coupling; Q, quadrupolar coupling.
Nature of the Sample Depending upon the kind of information desired, membrane bilayer samples can be prepared either oriented with respect to the applied field, or as random dispersions. For most studies, full hydration (430 wt% of water) is desired, especially for protein studies where denaturation may occur and biological function be lost without sufficient amounts of water present. Oriented Membranes Both natural and synthetic membranes can be effectively oriented and studied using NMR. In general, reducing the hydration level of biomembranes supported and oriented on a substratum (glass or mica plates) improves their orientation, but if less than limiting levels of hydration are used (Bo30 wt%), alterations may occur in the lipid phase from bilayer to isotropic or hexagonal phases. Fortunately, for all orientational studies of membranes, a good internal check for orientation can be made from the 31P NMR spectrum of the bilayer phospholipids, since the line positions in the spectra recorded at 01 and 901 are separated by 40–50 ppm (Figure 3). The average mosaic spread can then be estimated from the spectral line-width. Synthetic, model membranes, made from pure lipids, are best oriented on glass plates by drying down an organic solvent (usually CHCl3/MeOH) solution of the lipid at 1–5 mg mL1. Overloading the lipids onto a substratum generally produces less good orientation, and some lipids (notably zwitterionic ones such as phosphatidylcholines and phosphatidylethanolamines, both of which are major natural membrane components) orient better than others (anionic ones in particular), with cholesterol aiding orientation. Removal of solvent under high vacuum (o104 torr; 1 torr ¼ 133.322 Pa) is followed by hydration, either by dropping buffer or water onto the film, or by incubating in a controlled atmosphere. It is also possible to orient hydrated random dispersions of lipids and proteins by applying pressure to the glass plates. In contrast, natural membranes are most readily oriented on glass plates using the isopotential spin dry ultracentrifugation (ISDU) method which involves centrifugation of a membrane dispersion followed by partial dehydration. Membrane proteins prepared by solid-phase peptide synthesis or expression in bacteria can be reconstituted into lipids from detergents or organic solvents. The samples are placed on glass plates whose size and shape are determined by the radiofrequency coil with flat or square geometries for optimal filling factors in stationary experiments, or by the rotor for magic angle oriented sample spinning (MAOSS) NMR experiments. While most stationary experiments utilize samples arranged so
830
1.04
66
16
9
1000
Relative sensitivity
High resolution and powder spectra Chemical shift s T1
High resolution spectra Chemical shift s T1, T2
High resolution and powder spectra Chemical shift s T1 NOE
High resolution spectra Chemical shift T1 Dipolar couplings
Powder spectra Quadrupole splitting T1, T2
High resolution spectra Chemical shift, T1, T2
Measured parameters
Chemical shift is sensitive to positional isomers Order parameters can be obtained High sensitivity Measurable in cells and in dispersed lipids
Natural abundance Chemical shift anisotropy is sensitive to headgroup environment and phase properties of the bulk lipids Measurable in cells and in dispersed lipids Cost of labelling is low Can be incorporated in growth media Chemical shift sensitive to conformation
Direct determination of order parameters and bond vectors Measurable in cells and dispersed lipids T1 dominated by fast (ns) motions T2 dominated by slow (ms– ms) motions Low natural abundance Natural abundance T1 dominated by one mechanism
High sensitivity Natural abundance
Advantages
Properties, advantages and disadvantages of the commonly used nuclei in studies of membranes
Two factors contribute to the line shape, complicating the analysis High power proton decoupling is difficult May induce chemical perturbation compared to 1H
Need for selective fluorination
Low natural abundance, means of labelling required Overlapping resonance
Individual lipid classes cannot be resolved in mixed bilayer systems unless sonicated or MAS NMR is used
Need MAS NMR to resolve spectra Without selective enrichment, overlapping resonances
Reasonable spectra with small vesicles, micelles, high speed MAS or MAS of oriented bilayers Several relaxation mechanisms Overlapping resonances Need for selective deuteration Low sensitivity
Disadvantages
s, Chemical shift anisotropy; T1, spin–lattice relaxation time; T2, spin–spin relaxation time; NOE, nuclear Overhauser effect; MAS, magic angle spinning.
F
19
N
15
P
31
C
13
H
2
H
1
Nucleus
Table 2
Ordering properties of phospholipids
Labelling of proteins and peptides Structural and dynamic studies
Lipid asymmetry Phase properties
Dynamic properties of phospholipids Lipid asymmetry Ligand–protein interactions Distance measurements Quantitation of lipid composition
Ordering properties of phospholipids Dynamic properties of phospholipids
Dynamic properties Lipid diffusion
Common applications
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Figure 3 Orientational dependence of the 31P NMR spectra (at 36.4 MHz) of planar multi-bilayers of phosphatidylcholine, where d is the angle of the applied field with respect to the membrane normal. T ¼ 77 1C. Reproduced with permission from Seelig J and Gally HU (1976) Investigation of phosphatidylethanolamine bilayers by deuterium and phosphorus-31 nuclear magnetic resonance. Biochemistry 15: 5199–5204.
that the bilayer normal is perpendicular to the direction of the applied magnetic field, it is possible to perform orientational-dependent studies with the addition of a goniometer, which is usually rotated from the probe base without probe removal from the magnet. For MAOSS NMR experiments, the smallest diameter circular thin glass plates which can be handled are usually 4 mm, but for greater sensitivity, larger (up to 14 mm) rotor probes can be used to accommodate larger plates. Random Dispersions of Membranes
Figure 2 Typical spectra for various nuclei exploited in studies of lipids in bilayers. 1H, a spectrum of phosphatidylcholine multibilayers, recorded under magic angle oriented sample spinning (MAOSS) conditions to give rise to narrow (B9 Hz) spectral lines; 13 C, proton-decoupled spectrum of sonicated, small (diameters o50 nm) phospholipid vesicles; 2H, a typical deuterium NMR spherically averaged powder pattern for phospholipid bilayers deuterated in the choline head group, showing the way in which the quadrupole splitting (Dnq) is determined; 19F, spectrum of sonicated vesicles of lipids specifically 19F-labelled in the 120 position of both acyl chains; 31P, a static, spherically averaged powder pattern from mixed, cardiolipin, phosphatidylcholine and phosphatidylethanolamine bilayers showing the lack of spectral resolution of the three lipid types (upper spectrum), and under MAS conditions to resolve the three individual phospholipids (lower spectrum).
Extensively sonicated dispersions of pure lipids usually form small single bilayer vesicles. These samples have been used extensively for 13C NMR relaxation studies, which describe the motional properties of the various lipid groups, but have not been widely applied to proteins in membranes. Natural membrane fragments or vesicles, large (diameter 450 nm) liposomes, and hydrated unsonicated lipid dispersions are all indistinguishable (because of their slow tumbling) from the NMR perspective. They all give rise to anisotropically broadened spectral envelopes without the application of solid-state NMR techniques. For these systems, a spherically averaged powder pattern is observed which may be narrowed by molecular motion. Magic angle sample spinning (at a rate of or) narrows many of the spectral features, but some potential orientational information content may be lost at high spinning rates (orcCSA, D or Q; Table 1) where there are few spinning side bands to analyse.
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Experimentally, the amount of sample required is determined by the sensitivity of the nucleus to be observed. Typically, mM of the sample (5–10 mg of lipid; 0.5–1 mg of a small peptide; 1–4 mg of a large protein) in a volume of about 0.7 mL or on 10–50 glass plates may be needed for less sensitive (2H, 15N) nuclei. For 1H, 19F, 31P and 13C in MAS NMR, somewhat less material is required. Cross-polarization from the abundant proton magnetization may improve sensitivity, and decoupling (5–10 kHz for 31P, 80–100 kHz for 1H) is routinely used to improve spectral shape and reduce spectral widths. Micelles and Bicelles Micelles (SDS is often used) and bicelles (made of long and short chain phosphatidylcholines) tumble isotropically in solution and can accommodate peptides and proteins (Figure 1) to provide better mimetics for membrane proteins and peptides than organic solvents, which themselves can induce secondary structures in peptides and proteins. Indeed, good protein function and high resolution NMR spectra can be obtained from micellar-protein complexes, with sufficient resolution to permit structural analysis. Bicelles align in the applied field with the membrane normal perpendicular to the applied field, and by doping the system with lanthanides (Tm, Yb, Er or Eu) the orientation can be turned through 901. Using paramagnetic chelates, direct protein–lanthanide interactions can be abolished leading to better spectral resolution although some hysteresis effects due to molecular reorganization may complicate their use.
Information Content Macroscopic Structures Phospholipids can, in aqueous dispersion, form a range of macroscopic structures including bilayers (predominantly for long chain derivatives, C12–C24), hexagonal, cubic and rhombic structures (Figure 4). 31P NMR of static samples is a good diagnostic way of identifying such structures. Spherically averaged powder patterns (with a CSA of B40–50 ppm) from bilayers are reversed and reduced in spectral widths (by 0.5) for hexagonal structures because of the added degree of freedom of molecular motion along the hexagonal cylinder. Isotropic spectra arise from small (diameter Z50 nm) vesicles, cubic, rhombic and micellar molecular arrangements, as a result of molecular reorientation of the structure with respect to the applied field which is fast enough to average the frequency-dependent 31P chemical shift anisotropy (tco3–10 kHz B ¼ 40–50 ppm on 200– 600 MHz instruments). Similar spectral changes are observed with 2H NMR for deuterated lipids in similar structures.
Figure 4 Three different types of phospholipid phases and their corresponding 31P NMR spectra. From Cullis PR and Kruijff B (1979) Lipid polymorphism and the functional roles of lipids in biological membranes. Biochimica et Biophysica Acta 559: 399–420.
A two-component bilayer and isotropic 31P NMR spectrum from a membrane (usually a natural membrane) has been interpreted in terms of a major bilayer structure encompassing a much smaller (r5%) population of inverted micelles. Although not the only explanation, the interpretation has many functionally attractive features (enhanced permeability, sites of membrane fusion, flip-flop regions, etc.). Such isotropic spectral components are often produced by proteins interacting with the surface of lipid bilayers. The identity of the lipid type, in a mixed lipid membrane, which exists in this isotropic environment, can be determined using MAS 31P NMR methods. Molecular Structure Lipid bilayers are two-dimensional, axially symmetric structures with the normal to the plane of the bilayer being a reference axis. Lipids usually rotate quickly (trBns) around their long axis in fluid bilayers, but peptides and proteins may lack this motion which is determined by the association (controlled oligomerization or irreversible aggregation) behaviour of the various components, as well as the lipid dynamics. Lipid orientational information
By exploiting the anisotropic properties of the nuclear spin-interactions of 2H, 13C and 15N (Table 1), placed at specific positions in peptides and ligand, rather precise bond vectors have been determined in oriented membranes.
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Deuterium has low natural abundance and a low g, but the quadrupole coupling can give rise to large splittings in the 2H NMR spectrum (Dnq(max) B 127 kHz), which is averaged by rotation of a methyl group around a C3 axis (to 127/3 B 40 kHz). This high degree of orientational sensitivity has been exploited to determine the structure of specific residues (valines) involved in dimeric association for the gramicidin ion channel, and the structure of retinal within its binding site of bacteriorhodopsin and rhodopsin. Spectral simulations are necessary, as is some estimate of line broadening due to macroscopic mosaic spread (from 31P spectral widths), but as a general method, the information gained is ab initio and, as such, model-independent. Most amino acids are available in a 2H-labelled form for incorporation into peptides in solid phase synthesis, and a wide range of 2H-precursors are available for specific labelling of ligands and prosthetic groups. Peptide and protein orientation
Many results have been obtained with specific, selective and uniform labelling of polypeptide sites with the spin I ¼ 1/2 nuclei 13C and 15N. These results have included the structure determination of gramicidin in bilayers, the geometrical arrangements of a variety of helical peptides in bilayers, the three-dimensional structures of peptides in bilayers, and the orientations of bound ligands and prosthetic groups. Uniform labelling of proteins with 15N offers many advantages, and, indeed, was first developed for solid-state NMR spectroscopy before being applied to solution-state NMR where it has achieved nearly universal use. The nitrogen sites in the protein backbone are separated by two carbon atoms, leaving only minimal homonuclear 15N dipolar effects, which is the essence of dilute-spin solid-state NMR spectroscopy. Each residue has one amide nitrogen in the backbone (with the exception of proline) and some have distinctive sidechain nitrogen sites. Further, uniform labelling allows the use of expressed proteins, and shifts the burden from sample preparation to spectroscopy, where complete spectral resolution is the essential starting point for structure determination. Multidimensional solid-state NMR experiments have been shown to yield completely resolved spectra of uniformly 15N labelled proteins in oriented lipid bilayers. In three-dimensional spectra, each amide resonance is characterized by three frequencies (1H chemical shift, 15 N chemical shift and 1H–15N heteronuclear dipolar coupling), which provide the source of resolution among the various sites as well as the basic input for structure determination based on orientational constraints. The data shown in Figure 5 are from a 50-residue protein in oriented lipid bilayers. More importantly, since the polypeptides are immobilized by the lipids on the relevant NMR time-scales, there can be no further
Figure 5 Multidimensional solid-state NMR spectra of uniformly 15N labelled fd coat protein in oriented lipid bilayers. Panel (a) is the complete two-dimensional 1H–15N heteronuclear dipolar–15N chemical shift spectrum. Panels (b) and (c) are spectral planes extracted from a three-dimensional correlation spectrum at specific 1H chemical shift frequencies. The spectral regions in the planes correspond to the boxed regions of the complete two-dimensional spectrum with which they are aligned. Panel (b) contains resonances with 1H chemical shift of 11.0 ppm. Panel (c) contains resonances with 1H chemical shift of 11.6 ppm. The three orientationally dependent frequencies that can be measured for all of the resonances in the threedimensional data set provide the input for structure determination. The arrow in panel (b) points to the resonance assigned to the amide of Leu 41 in the trans-membrane hydrophobic helix and that in panel (c) to the amide of Leu 14 in the in-plane amphipathic helix.
degradation of line widths or other spectroscopic properties as the size of the polypeptide increases. Although larger proteins will have more complex spectra resulting from the increased number of resonances, there is no fundamental size limitation to solid-state NMR studies of membrane proteins. In principle, a single three-dimensional correlation spectrum of an oriented sample of a uniformly 15N labelled protein provides sufficient information in the form of orientationally dependent frequencies for each amide site to determine the complete structure of the polypeptide backbone. The two-dimensional 1H–15N heteronuclear dipolar–15N chemical shift PISEMA spectrum in Figure 5a was obtained from a uniformly 15 N labelled sample of the 50-residue fd coat protein in oriented bilayers; it contains resonances from all of the amide backbone (and side-chain) nitrogen sites. The resonances in the box in the upper left are largely from residues in the trans-membrane hydrophobic a-helix, and the resonances in the box on the right are from residues in the in-plane amphipathic a-helix. The two-dimensional PISEMA spectrum is generally well resolved, although there is some overlap among the resonances from residues in the trans-membrane a-helix because their peptide bonds have similar orientations, approximately parallel to the magnetic
Membranes Studied by NMR Spectroscopy
field. Two-dimensional planes extracted from a threedimensional correlation spectrum of the same sample are shown in Figure 5b and 5c. The spectral regions in the planes correspond to the boxed regions of the complete two-dimensional spectrum with which they are aligned. There are very few resonances in the spectral regions in Figure 5b and 5c because only those resonances with a specific 1H chemical shift, the third frequency in the three-dimensional spectrum, appear in each plane. These data illustrate how the three-dimensional correlation experiment contributes to these studies. First, it provides a substantial increase in resolution by separating resonances based on their 1H chemical shift frequencies. Second, it enables the direct measurement of three orientationally dependent frequencies for each resolved resonances, the 1H chemical shift, 1 H–15N heteronuclear dipolar coupling and the 15N chemical shift. Because the orientation of the proteincontaining bilayers is fixed by the method of sample preparation, these frequencies can be used to determine the orientation of each peptide plane with respect to the applied magnetic field, since the magnitudes and orientations of the spin-interaction tensors for the amide sites in the molecular frame are known. Structures are then determined using the frequencies associated with individual resonances that have been assigned to specific residues as angular constraints. Using this approach the three-dimensional structure of the M2 trans-membrane segment from the acetylcholine receptor in oriented bilayers was determined. The 13C chemical shift and 2H quadrupolar coupling frequencies measured from selectively or specifically labelled samples in separate experiments also provide valuable structural information, and have been used together with the 15N chemical shift and the 1H–15N heteronuclear dipolar coupling to determine the structure of the gramicidin channel at high resolution.
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where (e2Qq/h) ¼ 127 kHz and thus jS j ¼
ðDnq Þobs ðDnq Þmax
and so 14|S|40. The order parameter profile (S at various positions of measurement) usually observed for lipid acyl chains displays a plateau for lipid methylenes in the upper part of the bilayer (C2–C10; SB0.4) as a result of water penetration and ordering of the upper part of the bilayer and then a decrease to the centre of the membrane (C10–C16 gives S values of 0.4–0.1) (Figure 6). The discontinuity in S-profile at C10–C12 corresponds to the main position of unsaturation in natural membranes. At this position, the static kink in the chain contributes geometrically to the angle about which motional averaging occurs, thereby reducing the molecular order
Amplitude of motion–order parameters
Partial but fast (tcoDnQ(max); D CSA(max)) motional averaging of spectral anisotropy permits an order parameter, and hence an angle of motional amplitude with respect to a fixed axis (the membrane normal), to be determined. Direct measurements of order parameters from the powder patterns of random bilayer dispersions of deuterated lipids are conveniently made from the measured quadrupole splittings (Dnq) (Figure 2), determined from the spectral maxima, corresponding to the 901-orientational spectral components. Thus: Dnq ¼
e 2 Qq h
3cos 2 y 1
Figure 6 The measured deuterium order parameters, SCD for the sn-1 (saturated) and sn-2 (unsaturated, C9 ¼ C10) bilayers of phospholipids specifically deuterated in their acyl chains, showing the consequence of the presence of double bonds causing the staggered conformation of the chains thus affecting the quadrupolar averaging (b), even though the molecular order parameter, Smol, remains for each chain (a). Reproduced with permission from Seelig J and Seelig A (1977) Effect of single cis double bond on the structure of a phospholipid bilayer. Biochemistry 16: 45–50.
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Figure 7 Deuterium NMR quadrupole splittings (Dnq) of the b-CD2 group plotted against the corresponding splitting for the a-CD2 group of dipalmitoylphosphatidylcholine with -P(O4)– aCD2–bCD2–Nþ(CH3)3 in bilayers for a range of ions at constant ionic strength, I ¼ 1.05 M and at 59 1C, showing the sensitivity of lipid head group orientation to surface charge. Reproduced with permission from Akutsu H and Seelig J (1981) Interaction of metal ions with phosphotidylcholine bilayer membranes. Biochemistry 20: 7366–7370.
parameter (Smol determined from Dnq(obs)), since: jSmol j ¼ jSðorientationÞj jSðamplitudeÞj
Labelled 2H lipid head groups give rise to small (Dnqo 10 kHz) quadrupole splitting, since their amplitude of motion with respect to the membrane normal is large, and their axis of averaging is not the membrane normal. In this case, the angle of molecular averaging cannot be uniquely determined, but the experimentally determined DnQ values can be useful in studying ion binding (Figure 7), peptide and protein interactions, and electrostatic interactions, since the phospholipid head group acts as a molecular voltmeter and sensor of interfacial pH at the bilayer surface.
Distance measurements within membranes
In MAS NMR, sample spinning averages out the dipolar couplings which are required for distance determinations. However, internuclear distance measurements can be made using rotational resonance (R2) for homonuclear spins and REDOR for heteronuclear spins, in which the dipolar couplings are reintroduced into the spectrum under special spinning conditions. By spinning (at a speed of or) the sample at multiples (n, where n ¼ 1, 2, 3, 4y) of the chemical shift difference (D in Hz) between a specific spin pair such that or ¼ nD, then transfer of magnetization occurs between the spin pair, and the dipolar interactions
are recoupled. Now, the dependence of the NMR spectral intensity with mixing time shows a dependence on the distance between the spin pairs, and hence the internuclear distance (r) can be determined. This approach has been used to determine 13C spin pair distances to sub-nm resolution in membranes between neighbouring lipids, between lipids and proteins (Figure 8), within a ligand at informative sites to give the structure of ligand at its site of action in a membrane bound target and of a prosthetic group (retinal) in its binding site of a receptor. Paramagnetic spectral broadening for a MAS NMR observed phosphate induced by a nitroxide spin-label, both at known positions in the primary sequence of membrane-bound bovine rhodopsin, a photoreceptor, has permitted some estimate of helix-loop distances to be made in the protein, supplementing other indirect structural details, even though the protein crystal structure is not available. Membrane Dynamics Both spin–lattice (T1) and spin–spin (T2) relaxation times give motional information about specific nuclei in a membrane system. Faster (tcB107–109 s) motions associated with acyl chain trans–gauche rotational isomerisms and protein residue motions affect T1 values, whereas slower (tcB103–104 s) peptide backbone and membrane director fluctuations are inherently detected by T2 values. Conventionally, high resolution NMR methods for measuring these relaxation times are used, the only difference being that broad spectral envelopes may show anisotropy in relaxation characteristics. Membrane protein side chains (for example, Lys-eCH2; Val-gCH3) have been shown to possess fast (ms) motions from T1 measurements of specifically deuterated residues in bacteriorhodopsin, even though the membrane environment was relatively rigid and crystalline. Similar approaches showed that the a-CH3 group rotation is fast (tco oms) for membrane-bound, specifically deuterated retinal in the same protein, even at 60 1C. Somewhat slower motions (in the ms range) occur in peptide backbones of membrane proteins. An enhancement of the T1 for cardiolipin phosphates through direct contact of the haem group in cytochrome c, a peripherally bound protein, suggests that this protein undergoes considerable conformational distortions (molten globule) when at the bilayer surface, on the 106 s time-scale, a feature of peripherally bound proteins which may be general. Slow (ms) director fluctuations (wobbling around the membrane normal) are induced in bilayer membranes by proteins, as shown by measurements of T2. These motions are strongly coupled through the protein and membrane and may be significant in maintaining the protein in a dynamic equilibrium ready for function.
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Figure 8 Rotational resonance 13C MAS NMR has been used to determine both an intramolecular distance within a lipid and an intermolecular distance between a lipid and a protein in bilayers. A train of spectra are shown at different mixing times for the n ¼ 2 resonance condition at a spinning speed of 624875 Hz, at two different temperatures, giving a distance (from an analysis of the intensity ˚ at 50 1C in changes with mixing time) from the C1 on the sn-1 chain to the C2 on the sn-2 chain of 4.0–5.0 A dipalmitoylphosphatidylcholine bilayers (a). To determine the intermolecular peptide–lipid distance, the spectral intensity changes due to magnetization transfer were determined under conditions of no magnetization transfer, that is, at off-resonance, (&), with unlabelled lipid to show the contribution from natural abundance (1.1%) 13C, (’), and with 13C-1,2-[2-13C] labelled lipid in bilayers containing ˚ . Reproduced with permission from Smith SO, glycophorin labelled at residue, 13C–OH tyrosine-93 (), to give a distance of 4.0–5.0A Hamilton J, Salmon A, and Bormann BJ (1994) Rotational resonance NMR determination of intra- and intermolecular distance constraints in dipalmitoylphosphatidylcholine bilayers. Biochemistry 33: 6327–6333.
Ligand Structure Small molecules activate a range of cellular responses following binding to membrane-bound receptors. Solidstate NMR methods permit the structure and binding kinetics of such small ligands, using isotropic labelling to aid assignment and enhance sensitivity. The b-ionone ring orientation (6-S trans or 6-S cis) of 13C-retinal in bacteriorhodopsin has been determined from direct
measurements using rotational resonance MAS NMR, by comparison with the known crystal structure of retinal. Deuterated retinal in bacteriorhodopsin and mammalian rhodopsin has been observed and the structural changes induced upon light incidence determined to good precision using an ab initio approach. 13C-labelled retinal has proved particularly successful for use in describing sugar binding to sugar transporters, drug binding to P-type ATPases (Hþ/Kþ-ATPase and Naþ/Kþ-ATPase) and
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acetylcholine binding to the membrane-bound receptor. In the absence of crystals of this class of proteins at the present time, detailed structural information from solidstate NMR approaches and observation of a range of isotopes are helping to elucidate functional descriptions by this approach. See also: 13C NMR, Methods, 13C NMR, Parameter Survey, Cells Studied by NMR, Diffusion Studied Using NMR Spectroscopy, Liquid Crystals and Liquid Crystal Solutions Studied by NMR, Macromolecule–Ligand Interactions Studied by NMR, NMR in Anisotropic Systems, Theory, 31P NMR, Solid State NMR, Methods, Solid State NMR, Rotational Resonance, Solid State NMR Using Quadrupolar Nuclei.
Further Reading Cross TA and Opella SJ (1994) Solid-state NMR structural studies of peptides and proteins in membranes. Current Opinion in Structural Biology 4: 574--581. Glaubitz C and Watts A (1998) Magic angle-oriented sample spinning (MAOSS): A new approach toward biomembrane studies. Journal of Magnetic Resonance 130: 305--316. Gro¨bner G, Taylor A, Williamson PTF, et al. (1997) Macroscopic orientation of natural and model membranes for structural studies. Analytical Biochemistry 254: 132--136. Marassi FM, Ramamoorthy A, and Opella SJ (1997) Complete resolution of the solid-sate NMR spectrum of a uniformly 15N-labeled membrane protein in phospholipid bilayers. Proceedings of the National Academy of Sciences 94: 8551--8556. Opella SJ (1997) NMR and membrane proteins. Nature Structural Biology 4(suppl.): 845--848.
Opella SJ, Stewart PL, and Valentine KG (1987) Protein structure by solid-state NMR spectroscopy. Quarterly Review of Biophysics 19: 7--49. Opella SJ, Marassi FM, Gesell JJ, et al. (1999) Three-dimensional structure of the membrane embedded M2 channel-lining segment from nicotinic acetylcholine receptors and NMDA receptors by NMR spectroscopy. Nature Structural Biology 6: 374--379. Pines A, Gibby MG, and Waugh JS (1973) Proton-enhanced nmr of dilute spins in solids. Journal of Chemical Physics 59: 569--590. Pinheiro TJT and Watts A (1994) Resolution of individual lipids in mixed phospholipid membranes and specific lipid-cytochrome c interactions by magic angle spinning solid-state phosphorus-31 NMR. Biochemistry 33: 2459--2467. Ramamoorthy A, Marassi FM, and Opella SJ (1996) Applications of multidimensional solid-state NMR spectroscopy to membrane proteins. In: Jardetzky O and Lefevre J (eds.) Dynamics and the Problem of Recognition in Biological Macromolecules, pp. 237--255. New York: Plenum. Reid DG (ed.) (1997) Protein NMR techniques In: Methods in Molecular Biology. Totawa, New Jersey: Humana Press. Sanders CR II and Landis GC (1995) Reconstitution of membrane proteins into lipid-rich bilayered mixed micelles for NMR studies. Biochemistry 34: 4030--4040. Smith SO, Ascheim K, and Groesbeck M (1996) Magic angle spinning NMR spectroscopy of membrane proteins. Quarterly Review of Biophysics 29: 395--449. Vold RR, Prosser RS, and Deese AJ (1997) Isotropic solutions of phospholipid bicelles: A new membrane mimetic for high-resolution NMR studies of polypeptides. Journal of Biomolecular NMR 9: 329--335. Watts A, Ulrich AS, and Middleton DA (1995) Membrane protein structure: The contribution and potential of novel solid state NMR approaches. Molecular Membrane Biology 12: 233--246. Watts A (1993) Magnetic resonance studies of phospholipid–protein interactions in bilayers. In: Cevc G (ed.) Phospholipids Handbook, pp. 687--740. New York: Marcel Dekker. Watts A (1998) Solid state NMR approaches for studying the interaction of peptides and proteins with membranes. Biochimica et Biophysica Acta 1376: 297--318. Watts A (1999) NMR of drugs and ligands bound to membrane receptors. Current Opinion in Biotechnology 10: 48--53.
Introduction Molecular motions in biomembranes are highly anisotropic. Since essentially all aspects of NMR spectroscopy are affected by molecular motions, the details of these motions strongly influence the spectroscopic approaches that can be applied to membrane samples. Although the motions of individual molecules or molecular groups may be very rapid (the molecular correlation time, tc, is in the ns range for lipid hydrocarbon chains and amino acid side chains of proteins), the overall tumbling of the lipid and polypeptide molecules in membrane bilayers is very slow and can be in the ms or s range. For this reason, conventional solution-state NMR, which relies on rapid overall molecular reorientation to narrow the resonance lines, has found little success in membrane research; even a lipid molecule with a relative molecular mass (Mr) of B1000 or a small peptide (MrB3000) has, when in membrane bilayers, by solution-state NMR criteria a tumbling rate equivalent to a very large (Mrc30 kDa) macromolecule in free solution. Only by sonicating the sample to produce small (diameter 20–50 nm) bilayer vesicles can conventional solution-state NMR methods be used. However, not only are there very serious concerns about the status of the lipids and polypeptides embedded in highly distorted lipid bilayers, but also those portions of proteins embedded in the bilayers may still reorient too slowly to yield resolvable resonances. Solution-state NMR spectroscopy has been applied to peptides and proteins in organic solvents, detergent micelles and bicelles (Figure 1), and structures have been determined in these model membrane environments. While micelles certainly provide a more relevant environment for membrane proteins than do mixtures of organic solvents, fully hydrated lipid bilayers remain the definitive environment for structural and functional studies of membrane proteins. As an alternative approach to solution-state NMR methods, which are ineffective with lipid bilayer samples, solid-state NMR methods have been refined sufficiently to permit structural details to be obtained for membraneembedded peptides and proteins. This usually requires isotopic enrichment, either through chemical synthesis or through biosynthetic incorporation in expressed peptides and proteins. In the absence of routine X-ray
Figure 1 Representation of the types of sample preparations used for studying membrane lipids and proteins. Micelles and bicelles are usually small (diameters B20–50 nm) structures and bilayers can be sonicated into small (20–50 nm diameter) vesicles, or produced as extended (diametersc100 nm) multibilayered or single bilayered closed or open structures, depending upon the method of preparation. Natural membranes are usually as large bilayer fragments or closed structures containing a complex and heterogeneous mixture of lipids and proteins and possibly carbohydrates.
crystallographic structural studies for these molecules, solid-state NMR spectroscopy has the potential to be a powerful and unique approach to determining the structures and describing the dynamics and functions of membranes and membrane-bound proteins. In addition, solid-state NMR spectroscopy has been widely used to describe lipid structure, dynamics and phase properties. Thus, solid-state NMR experiments can be applied to both the polypeptide and lipid components of membrane bilayers, without the need to disrupt the sample through sonication or the addition of organic solvents or detergents.
Nuclei Used in Membrane Studies With the exception of J-couplings, the major magnetic interactions (chemical shift, dipolar and quadrupolar couplings) for the nuclei exploited in biological NMR can be averaged with respect to the applied fields (B0 BMHz and B1BkHz) by isotropic molecular motion of small molecules (Table 1). However, for biomembranes, any of these interactions may yield resonances with very broad lines and dominate the spectra, masking the resolution required for high resolution studies. Where these
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interactions can be exploited, their anisotropy (usually chemical shift, dipolar or quadrupolar) can give molecular orientational information from static samples, either oriented or as random dispersions (see below). Alternatively, magic angle spinning (MAS) of the sample can be used at spinning speeds (or) which are either fast enough to average the interaction completely (orcs, D, Q) to give high resolution-like solid-state NMR spectra, or may be moderated either to recouple a dipolar interaction, such as in rotational resonance or REDOR, or to provide orientionally dependent spinning spectral side-bands for nuclei which display chemical shift anisotropy (e.g. 31P, 15N). Naturally occurring 13C (natural abundance and with selective enrichment) and 31P nuclei have been extensively exploited in membrane NMR studies (Table 2). However, replacement of 1H by 2H or 19F, and 14N by 15 N, has also found widespread application, although to date 17O has not found application in these systems. Typical spectra for the more commonly exploited nuclei for lipids in bilayers are shown in Figure 2. The need to average the strong dipolar coupling (B100 kHz) for 1H to obtain high resolution spectra has excluded widespread observation of this nucleus in membranes. Extensive protein deuteration, to leave a minor 1H density at a site of interest for observation in micellar suspensions, has been achieved. The realization that reorientation around the long molecular axis rotation of lipids and proteins in membrane bilayers in the liquid crystalline phase is sufficiently fast (at about 109 Hz for lipids and 106 Hz for proteins with radius r4 nm in fluid membranes) to average even homonuclear 1H dipolar couplings, has opened a new avenue for membrane studies for most observable nuclei, including 1H without the need for isotopic replacements. In addition, it is possible to perform magic angle oriented sample spinning (MAOSS) experiments to reap the benefits of both sample orientation and magic angle sample spinning in this situation.
Table 1 Comparison of the strengths of the magnetic interactions in NMR and the ways in which they can be averaged to yield molecular information for lipids, peptides and proteins in membranes in liquid state and solid state approaches Interaction
Liquids (Hz)
Solids (Hz)
Methods
s J D Q
10–104 102 0 0
10–104 102 104 105–106
MAS Decoupling Decoupling, MAS MAS
Adapted with permission from Smith SO, Ascheim K, and Groesbeck M (1996) Magic angle spinning NMR spectroscopy of membrane proteins. Quarterly Review of Biophysics 29: 395–449. s, Chemical shift anisotropy; J, J-coupling; D, dipolar coupling; Q, quadrupolar coupling.
Nature of the Sample Depending upon the kind of information desired, membrane bilayer samples can be prepared either oriented with respect to the applied field, or as random dispersions. For most studies, full hydration (430 wt% of water) is desired, especially for protein studies where denaturation may occur and biological function be lost without sufficient amounts of water present. Oriented Membranes Both natural and synthetic membranes can be effectively oriented and studied using NMR. In general, reducing the hydration level of biomembranes supported and oriented on a substratum (glass or mica plates) improves their orientation, but if less than limiting levels of hydration are used (Bo30 wt%), alterations may occur in the lipid phase from bilayer to isotropic or hexagonal phases. Fortunately, for all orientational studies of membranes, a good internal check for orientation can be made from the 31P NMR spectrum of the bilayer phospholipids, since the line positions in the spectra recorded at 01 and 901 are separated by 40–50 ppm (Figure 3). The average mosaic spread can then be estimated from the spectral line-width. Synthetic, model membranes, made from pure lipids, are best oriented on glass plates by drying down an organic solvent (usually CHCl3/MeOH) solution of the lipid at 1–5 mg mL1. Overloading the lipids onto a substratum generally produces less good orientation, and some lipids (notably zwitterionic ones such as phosphatidylcholines and phosphatidylethanolamines, both of which are major natural membrane components) orient better than others (anionic ones in particular), with cholesterol aiding orientation. Removal of solvent under high vacuum (o104 torr; 1 torr ¼ 133.322 Pa) is followed by hydration, either by dropping buffer or water onto the film, or by incubating in a controlled atmosphere. It is also possible to orient hydrated random dispersions of lipids and proteins by applying pressure to the glass plates. In contrast, natural membranes are most readily oriented on glass plates using the isopotential spin dry ultracentrifugation (ISDU) method which involves centrifugation of a membrane dispersion followed by partial dehydration. Membrane proteins prepared by solid-phase peptide synthesis or expression in bacteria can be reconstituted into lipids from detergents or organic solvents. The samples are placed on glass plates whose size and shape are determined by the radiofrequency coil with flat or square geometries for optimal filling factors in stationary experiments, or by the rotor for magic angle oriented sample spinning (MAOSS) NMR experiments. While most stationary experiments utilize samples arranged so
830
1.04
66
16
9
1000
Relative sensitivity
High resolution and powder spectra Chemical shift s T1
High resolution spectra Chemical shift s T1, T2
High resolution and powder spectra Chemical shift s T1 NOE
High resolution spectra Chemical shift T1 Dipolar couplings
Powder spectra Quadrupole splitting T1, T2
High resolution spectra Chemical shift, T1, T2
Measured parameters
Chemical shift is sensitive to positional isomers Order parameters can be obtained High sensitivity Measurable in cells and in dispersed lipids
Natural abundance Chemical shift anisotropy is sensitive to headgroup environment and phase properties of the bulk lipids Measurable in cells and in dispersed lipids Cost of labelling is low Can be incorporated in growth media Chemical shift sensitive to conformation
Direct determination of order parameters and bond vectors Measurable in cells and dispersed lipids T1 dominated by fast (ns) motions T2 dominated by slow (ms– ms) motions Low natural abundance Natural abundance T1 dominated by one mechanism
High sensitivity Natural abundance
Advantages
Properties, advantages and disadvantages of the commonly used nuclei in studies of membranes
Two factors contribute to the line shape, complicating the analysis High power proton decoupling is difficult May induce chemical perturbation compared to 1H
Need for selective fluorination
Low natural abundance, means of labelling required Overlapping resonance
Individual lipid classes cannot be resolved in mixed bilayer systems unless sonicated or MAS NMR is used
Need MAS NMR to resolve spectra Without selective enrichment, overlapping resonances
Reasonable spectra with small vesicles, micelles, high speed MAS or MAS of oriented bilayers Several relaxation mechanisms Overlapping resonances Need for selective deuteration Low sensitivity
Disadvantages
s, Chemical shift anisotropy; T1, spin–lattice relaxation time; T2, spin–spin relaxation time; NOE, nuclear Overhauser effect; MAS, magic angle spinning.
F
19
N
15
P
31
C
13
H
2
H
1
Nucleus
Table 2
Ordering properties of phospholipids
Labelling of proteins and peptides Structural and dynamic studies
Lipid asymmetry Phase properties
Dynamic properties of phospholipids Lipid asymmetry Ligand–protein interactions Distance measurements Quantitation of lipid composition
Ordering properties of phospholipids Dynamic properties of phospholipids
Dynamic properties Lipid diffusion
Common applications
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Figure 3 Orientational dependence of the 31P NMR spectra (at 36.4 MHz) of planar multi-bilayers of phosphatidylcholine, where d is the angle of the applied field with respect to the membrane normal. T ¼ 77 1C. Reproduced with permission from Seelig J and Gally HU (1976) Investigation of phosphatidylethanolamine bilayers by deuterium and phosphorus-31 nuclear magnetic resonance. Biochemistry 15: 5199–5204.
that the bilayer normal is perpendicular to the direction of the applied magnetic field, it is possible to perform orientational-dependent studies with the addition of a goniometer, which is usually rotated from the probe base without probe removal from the magnet. For MAOSS NMR experiments, the smallest diameter circular thin glass plates which can be handled are usually 4 mm, but for greater sensitivity, larger (up to 14 mm) rotor probes can be used to accommodate larger plates. Random Dispersions of Membranes
Figure 2 Typical spectra for various nuclei exploited in studies of lipids in bilayers. 1H, a spectrum of phosphatidylcholine multibilayers, recorded under magic angle oriented sample spinning (MAOSS) conditions to give rise to narrow (B9 Hz) spectral lines; 13 C, proton-decoupled spectrum of sonicated, small (diameters o50 nm) phospholipid vesicles; 2H, a typical deuterium NMR spherically averaged powder pattern for phospholipid bilayers deuterated in the choline head group, showing the way in which the quadrupole splitting (Dnq) is determined; 19F, spectrum of sonicated vesicles of lipids specifically 19F-labelled in the 120 position of both acyl chains; 31P, a static, spherically averaged powder pattern from mixed, cardiolipin, phosphatidylcholine and phosphatidylethanolamine bilayers showing the lack of spectral resolution of the three lipid types (upper spectrum), and under MAS conditions to resolve the three individual phospholipids (lower spectrum).
Extensively sonicated dispersions of pure lipids usually form small single bilayer vesicles. These samples have been used extensively for 13C NMR relaxation studies, which describe the motional properties of the various lipid groups, but have not been widely applied to proteins in membranes. Natural membrane fragments or vesicles, large (diameter 450 nm) liposomes, and hydrated unsonicated lipid dispersions are all indistinguishable (because of their slow tumbling) from the NMR perspective. They all give rise to anisotropically broadened spectral envelopes without the application of solid-state NMR techniques. For these systems, a spherically averaged powder pattern is observed which may be narrowed by molecular motion. Magic angle sample spinning (at a rate of or) narrows many of the spectral features, but some potential orientational information content may be lost at high spinning rates (orcCSA, D or Q; Table 1) where there are few spinning side bands to analyse.
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Experimentally, the amount of sample required is determined by the sensitivity of the nucleus to be observed. Typically, mM of the sample (5–10 mg of lipid; 0.5–1 mg of a small peptide; 1–4 mg of a large protein) in a volume of about 0.7 mL or on 10–50 glass plates may be needed for less sensitive (2H, 15N) nuclei. For 1H, 19F, 31P and 13C in MAS NMR, somewhat less material is required. Cross-polarization from the abundant proton magnetization may improve sensitivity, and decoupling (5–10 kHz for 31P, 80–100 kHz for 1H) is routinely used to improve spectral shape and reduce spectral widths. Micelles and Bicelles Micelles (SDS is often used) and bicelles (made of long and short chain phosphatidylcholines) tumble isotropically in solution and can accommodate peptides and proteins (Figure 1) to provide better mimetics for membrane proteins and peptides than organic solvents, which themselves can induce secondary structures in peptides and proteins. Indeed, good protein function and high resolution NMR spectra can be obtained from micellar-protein complexes, with sufficient resolution to permit structural analysis. Bicelles align in the applied field with the membrane normal perpendicular to the applied field, and by doping the system with lanthanides (Tm, Yb, Er or Eu) the orientation can be turned through 901. Using paramagnetic chelates, direct protein–lanthanide interactions can be abolished leading to better spectral resolution although some hysteresis effects due to molecular reorganization may complicate their use.
Information Content Macroscopic Structures Phospholipids can, in aqueous dispersion, form a range of macroscopic structures including bilayers (predominantly for long chain derivatives, C12–C24), hexagonal, cubic and rhombic structures (Figure 4). 31P NMR of static samples is a good diagnostic way of identifying such structures. Spherically averaged powder patterns (with a CSA of B40–50 ppm) from bilayers are reversed and reduced in spectral widths (by 0.5) for hexagonal structures because of the added degree of freedom of molecular motion along the hexagonal cylinder. Isotropic spectra arise from small (diameter Z50 nm) vesicles, cubic, rhombic and micellar molecular arrangements, as a result of molecular reorientation of the structure with respect to the applied field which is fast enough to average the frequency-dependent 31P chemical shift anisotropy (tco3–10 kHz B ¼ 40–50 ppm on 200– 600 MHz instruments). Similar spectral changes are observed with 2H NMR for deuterated lipids in similar structures.
Figure 4 Three different types of phospholipid phases and their corresponding 31P NMR spectra. From Cullis PR and Kruijff B (1979) Lipid polymorphism and the functional roles of lipids in biological membranes. Biochimica et Biophysica Acta 559: 399–420.
A two-component bilayer and isotropic 31P NMR spectrum from a membrane (usually a natural membrane) has been interpreted in terms of a major bilayer structure encompassing a much smaller (r5%) population of inverted micelles. Although not the only explanation, the interpretation has many functionally attractive features (enhanced permeability, sites of membrane fusion, flip-flop regions, etc.). Such isotropic spectral components are often produced by proteins interacting with the surface of lipid bilayers. The identity of the lipid type, in a mixed lipid membrane, which exists in this isotropic environment, can be determined using MAS 31P NMR methods. Molecular Structure Lipid bilayers are two-dimensional, axially symmetric structures with the normal to the plane of the bilayer being a reference axis. Lipids usually rotate quickly (trBns) around their long axis in fluid bilayers, but peptides and proteins may lack this motion which is determined by the association (controlled oligomerization or irreversible aggregation) behaviour of the various components, as well as the lipid dynamics. Lipid orientational information
By exploiting the anisotropic properties of the nuclear spin-interactions of 2H, 13C and 15N (Table 1), placed at specific positions in peptides and ligand, rather precise bond vectors have been determined in oriented membranes.
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Deuterium has low natural abundance and a low g, but the quadrupole coupling can give rise to large splittings in the 2H NMR spectrum (Dnq(max) B 127 kHz), which is averaged by rotation of a methyl group around a C3 axis (to 127/3 B 40 kHz). This high degree of orientational sensitivity has been exploited to determine the structure of specific residues (valines) involved in dimeric association for the gramicidin ion channel, and the structure of retinal within its binding site of bacteriorhodopsin and rhodopsin. Spectral simulations are necessary, as is some estimate of line broadening due to macroscopic mosaic spread (from 31P spectral widths), but as a general method, the information gained is ab initio and, as such, model-independent. Most amino acids are available in a 2H-labelled form for incorporation into peptides in solid phase synthesis, and a wide range of 2H-precursors are available for specific labelling of ligands and prosthetic groups. Peptide and protein orientation
Many results have been obtained with specific, selective and uniform labelling of polypeptide sites with the spin I ¼ 1/2 nuclei 13C and 15N. These results have included the structure determination of gramicidin in bilayers, the geometrical arrangements of a variety of helical peptides in bilayers, the three-dimensional structures of peptides in bilayers, and the orientations of bound ligands and prosthetic groups. Uniform labelling of proteins with 15N offers many advantages, and, indeed, was first developed for solid-state NMR spectroscopy before being applied to solution-state NMR where it has achieved nearly universal use. The nitrogen sites in the protein backbone are separated by two carbon atoms, leaving only minimal homonuclear 15N dipolar effects, which is the essence of dilute-spin solid-state NMR spectroscopy. Each residue has one amide nitrogen in the backbone (with the exception of proline) and some have distinctive sidechain nitrogen sites. Further, uniform labelling allows the use of expressed proteins, and shifts the burden from sample preparation to spectroscopy, where complete spectral resolution is the essential starting point for structure determination. Multidimensional solid-state NMR experiments have been shown to yield completely resolved spectra of uniformly 15N labelled proteins in oriented lipid bilayers. In three-dimensional spectra, each amide resonance is characterized by three frequencies (1H chemical shift, 15 N chemical shift and 1H–15N heteronuclear dipolar coupling), which provide the source of resolution among the various sites as well as the basic input for structure determination based on orientational constraints. The data shown in Figure 5 are from a 50-residue protein in oriented lipid bilayers. More importantly, since the polypeptides are immobilized by the lipids on the relevant NMR time-scales, there can be no further
Figure 5 Multidimensional solid-state NMR spectra of uniformly 15N labelled fd coat protein in oriented lipid bilayers. Panel (a) is the complete two-dimensional 1H–15N heteronuclear dipolar–15N chemical shift spectrum. Panels (b) and (c) are spectral planes extracted from a three-dimensional correlation spectrum at specific 1H chemical shift frequencies. The spectral regions in the planes correspond to the boxed regions of the complete two-dimensional spectrum with which they are aligned. Panel (b) contains resonances with 1H chemical shift of 11.0 ppm. Panel (c) contains resonances with 1H chemical shift of 11.6 ppm. The three orientationally dependent frequencies that can be measured for all of the resonances in the threedimensional data set provide the input for structure determination. The arrow in panel (b) points to the resonance assigned to the amide of Leu 41 in the trans-membrane hydrophobic helix and that in panel (c) to the amide of Leu 14 in the in-plane amphipathic helix.
degradation of line widths or other spectroscopic properties as the size of the polypeptide increases. Although larger proteins will have more complex spectra resulting from the increased number of resonances, there is no fundamental size limitation to solid-state NMR studies of membrane proteins. In principle, a single three-dimensional correlation spectrum of an oriented sample of a uniformly 15N labelled protein provides sufficient information in the form of orientationally dependent frequencies for each amide site to determine the complete structure of the polypeptide backbone. The two-dimensional 1H–15N heteronuclear dipolar–15N chemical shift PISEMA spectrum in Figure 5a was obtained from a uniformly 15 N labelled sample of the 50-residue fd coat protein in oriented bilayers; it contains resonances from all of the amide backbone (and side-chain) nitrogen sites. The resonances in the box in the upper left are largely from residues in the trans-membrane hydrophobic a-helix, and the resonances in the box on the right are from residues in the in-plane amphipathic a-helix. The two-dimensional PISEMA spectrum is generally well resolved, although there is some overlap among the resonances from residues in the trans-membrane a-helix because their peptide bonds have similar orientations, approximately parallel to the magnetic
Membranes Studied by NMR Spectroscopy
field. Two-dimensional planes extracted from a threedimensional correlation spectrum of the same sample are shown in Figure 5b and 5c. The spectral regions in the planes correspond to the boxed regions of the complete two-dimensional spectrum with which they are aligned. There are very few resonances in the spectral regions in Figure 5b and 5c because only those resonances with a specific 1H chemical shift, the third frequency in the three-dimensional spectrum, appear in each plane. These data illustrate how the three-dimensional correlation experiment contributes to these studies. First, it provides a substantial increase in resolution by separating resonances based on their 1H chemical shift frequencies. Second, it enables the direct measurement of three orientationally dependent frequencies for each resolved resonances, the 1H chemical shift, 1 H–15N heteronuclear dipolar coupling and the 15N chemical shift. Because the orientation of the proteincontaining bilayers is fixed by the method of sample preparation, these frequencies can be used to determine the orientation of each peptide plane with respect to the applied magnetic field, since the magnitudes and orientations of the spin-interaction tensors for the amide sites in the molecular frame are known. Structures are then determined using the frequencies associated with individual resonances that have been assigned to specific residues as angular constraints. Using this approach the three-dimensional structure of the M2 trans-membrane segment from the acetylcholine receptor in oriented bilayers was determined. The 13C chemical shift and 2H quadrupolar coupling frequencies measured from selectively or specifically labelled samples in separate experiments also provide valuable structural information, and have been used together with the 15N chemical shift and the 1H–15N heteronuclear dipolar coupling to determine the structure of the gramicidin channel at high resolution.
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where (e2Qq/h) ¼ 127 kHz and thus jS j ¼
ðDnq Þobs ðDnq Þmax
and so 14|S|40. The order parameter profile (S at various positions of measurement) usually observed for lipid acyl chains displays a plateau for lipid methylenes in the upper part of the bilayer (C2–C10; SB0.4) as a result of water penetration and ordering of the upper part of the bilayer and then a decrease to the centre of the membrane (C10–C16 gives S values of 0.4–0.1) (Figure 6). The discontinuity in S-profile at C10–C12 corresponds to the main position of unsaturation in natural membranes. At this position, the static kink in the chain contributes geometrically to the angle about which motional averaging occurs, thereby reducing the molecular order
Amplitude of motion–order parameters
Partial but fast (tcoDnQ(max); D CSA(max)) motional averaging of spectral anisotropy permits an order parameter, and hence an angle of motional amplitude with respect to a fixed axis (the membrane normal), to be determined. Direct measurements of order parameters from the powder patterns of random bilayer dispersions of deuterated lipids are conveniently made from the measured quadrupole splittings (Dnq) (Figure 2), determined from the spectral maxima, corresponding to the 901-orientational spectral components. Thus: Dnq ¼
e 2 Qq h
3cos 2 y 1
Figure 6 The measured deuterium order parameters, SCD for the sn-1 (saturated) and sn-2 (unsaturated, C9 ¼ C10) bilayers of phospholipids specifically deuterated in their acyl chains, showing the consequence of the presence of double bonds causing the staggered conformation of the chains thus affecting the quadrupolar averaging (b), even though the molecular order parameter, Smol, remains for each chain (a). Reproduced with permission from Seelig J and Seelig A (1977) Effect of single cis double bond on the structure of a phospholipid bilayer. Biochemistry 16: 45–50.
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Figure 7 Deuterium NMR quadrupole splittings (Dnq) of the b-CD2 group plotted against the corresponding splitting for the a-CD2 group of dipalmitoylphosphatidylcholine with -P(O4)– aCD2–bCD2–Nþ(CH3)3 in bilayers for a range of ions at constant ionic strength, I ¼ 1.05 M and at 59 1C, showing the sensitivity of lipid head group orientation to surface charge. Reproduced with permission from Akutsu H and Seelig J (1981) Interaction of metal ions with phosphotidylcholine bilayer membranes. Biochemistry 20: 7366–7370.
parameter (Smol determined from Dnq(obs)), since: jSmol j ¼ jSðorientationÞj jSðamplitudeÞj
Labelled 2H lipid head groups give rise to small (Dnqo 10 kHz) quadrupole splitting, since their amplitude of motion with respect to the membrane normal is large, and their axis of averaging is not the membrane normal. In this case, the angle of molecular averaging cannot be uniquely determined, but the experimentally determined DnQ values can be useful in studying ion binding (Figure 7), peptide and protein interactions, and electrostatic interactions, since the phospholipid head group acts as a molecular voltmeter and sensor of interfacial pH at the bilayer surface.
Distance measurements within membranes
In MAS NMR, sample spinning averages out the dipolar couplings which are required for distance determinations. However, internuclear distance measurements can be made using rotational resonance (R2) for homonuclear spins and REDOR for heteronuclear spins, in which the dipolar couplings are reintroduced into the spectrum under special spinning conditions. By spinning (at a speed of or) the sample at multiples (n, where n ¼ 1, 2, 3, 4y) of the chemical shift difference (D in Hz) between a specific spin pair such that or ¼ nD, then transfer of magnetization occurs between the spin pair, and the dipolar interactions
are recoupled. Now, the dependence of the NMR spectral intensity with mixing time shows a dependence on the distance between the spin pairs, and hence the internuclear distance (r) can be determined. This approach has been used to determine 13C spin pair distances to sub-nm resolution in membranes between neighbouring lipids, between lipids and proteins (Figure 8), within a ligand at informative sites to give the structure of ligand at its site of action in a membrane bound target and of a prosthetic group (retinal) in its binding site of a receptor. Paramagnetic spectral broadening for a MAS NMR observed phosphate induced by a nitroxide spin-label, both at known positions in the primary sequence of membrane-bound bovine rhodopsin, a photoreceptor, has permitted some estimate of helix-loop distances to be made in the protein, supplementing other indirect structural details, even though the protein crystal structure is not available. Membrane Dynamics Both spin–lattice (T1) and spin–spin (T2) relaxation times give motional information about specific nuclei in a membrane system. Faster (tcB107–109 s) motions associated with acyl chain trans–gauche rotational isomerisms and protein residue motions affect T1 values, whereas slower (tcB103–104 s) peptide backbone and membrane director fluctuations are inherently detected by T2 values. Conventionally, high resolution NMR methods for measuring these relaxation times are used, the only difference being that broad spectral envelopes may show anisotropy in relaxation characteristics. Membrane protein side chains (for example, Lys-eCH2; Val-gCH3) have been shown to possess fast (ms) motions from T1 measurements of specifically deuterated residues in bacteriorhodopsin, even though the membrane environment was relatively rigid and crystalline. Similar approaches showed that the a-CH3 group rotation is fast (tco oms) for membrane-bound, specifically deuterated retinal in the same protein, even at 60 1C. Somewhat slower motions (in the ms range) occur in peptide backbones of membrane proteins. An enhancement of the T1 for cardiolipin phosphates through direct contact of the haem group in cytochrome c, a peripherally bound protein, suggests that this protein undergoes considerable conformational distortions (molten globule) when at the bilayer surface, on the 106 s time-scale, a feature of peripherally bound proteins which may be general. Slow (ms) director fluctuations (wobbling around the membrane normal) are induced in bilayer membranes by proteins, as shown by measurements of T2. These motions are strongly coupled through the protein and membrane and may be significant in maintaining the protein in a dynamic equilibrium ready for function.
Membranes Studied by NMR Spectroscopy
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Figure 8 Rotational resonance 13C MAS NMR has been used to determine both an intramolecular distance within a lipid and an intermolecular distance between a lipid and a protein in bilayers. A train of spectra are shown at different mixing times for the n ¼ 2 resonance condition at a spinning speed of 624875 Hz, at two different temperatures, giving a distance (from an analysis of the intensity ˚ at 50 1C in changes with mixing time) from the C1 on the sn-1 chain to the C2 on the sn-2 chain of 4.0–5.0 A dipalmitoylphosphatidylcholine bilayers (a). To determine the intermolecular peptide–lipid distance, the spectral intensity changes due to magnetization transfer were determined under conditions of no magnetization transfer, that is, at off-resonance, (&), with unlabelled lipid to show the contribution from natural abundance (1.1%) 13C, (’), and with 13C-1,2-[2-13C] labelled lipid in bilayers containing ˚ . Reproduced with permission from Smith SO, glycophorin labelled at residue, 13C–OH tyrosine-93 (), to give a distance of 4.0–5.0A Hamilton J, Salmon A, and Bormann BJ (1994) Rotational resonance NMR determination of intra- and intermolecular distance constraints in dipalmitoylphosphatidylcholine bilayers. Biochemistry 33: 6327–6333.
Ligand Structure Small molecules activate a range of cellular responses following binding to membrane-bound receptors. Solidstate NMR methods permit the structure and binding kinetics of such small ligands, using isotropic labelling to aid assignment and enhance sensitivity. The b-ionone ring orientation (6-S trans or 6-S cis) of 13C-retinal in bacteriorhodopsin has been determined from direct
measurements using rotational resonance MAS NMR, by comparison with the known crystal structure of retinal. Deuterated retinal in bacteriorhodopsin and mammalian rhodopsin has been observed and the structural changes induced upon light incidence determined to good precision using an ab initio approach. 13C-labelled retinal has proved particularly successful for use in describing sugar binding to sugar transporters, drug binding to P-type ATPases (Hþ/Kþ-ATPase and Naþ/Kþ-ATPase) and
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acetylcholine binding to the membrane-bound receptor. In the absence of crystals of this class of proteins at the present time, detailed structural information from solidstate NMR approaches and observation of a range of isotopes are helping to elucidate functional descriptions by this approach. See also: 13C NMR, Methods, 13C NMR, Parameter Survey, Cells Studied by NMR, Diffusion Studied Using NMR Spectroscopy, Liquid Crystals and Liquid Crystal Solutions Studied by NMR, Macromolecule–Ligand Interactions Studied by NMR, NMR in Anisotropic Systems, Theory, 31P NMR, Solid State NMR, Methods, Solid State NMR, Rotational Resonance, Solid State NMR Using Quadrupolar Nuclei.
Further Reading Cross TA and Opella SJ (1994) Solid-state NMR structural studies of peptides and proteins in membranes. Current Opinion in Structural Biology 4: 574--581. Glaubitz C and Watts A (1998) Magic angle-oriented sample spinning (MAOSS): A new approach toward biomembrane studies. Journal of Magnetic Resonance 130: 305--316. Gro¨bner G, Taylor A, Williamson PTF, et al. (1997) Macroscopic orientation of natural and model membranes for structural studies. Analytical Biochemistry 254: 132--136. Marassi FM, Ramamoorthy A, and Opella SJ (1997) Complete resolution of the solid-sate NMR spectrum of a uniformly 15N-labeled membrane protein in phospholipid bilayers. Proceedings of the National Academy of Sciences 94: 8551--8556. Opella SJ (1997) NMR and membrane proteins. Nature Structural Biology 4(suppl.): 845--848.
Opella SJ, Stewart PL, and Valentine KG (1987) Protein structure by solid-state NMR spectroscopy. Quarterly Review of Biophysics 19: 7--49. Opella SJ, Marassi FM, Gesell JJ, et al. (1999) Three-dimensional structure of the membrane embedded M2 channel-lining segment from nicotinic acetylcholine receptors and NMDA receptors by NMR spectroscopy. Nature Structural Biology 6: 374--379. Pines A, Gibby MG, and Waugh JS (1973) Proton-enhanced nmr of dilute spins in solids. Journal of Chemical Physics 59: 569--590. Pinheiro TJT and Watts A (1994) Resolution of individual lipids in mixed phospholipid membranes and specific lipid-cytochrome c interactions by magic angle spinning solid-state phosphorus-31 NMR. Biochemistry 33: 2459--2467. Ramamoorthy A, Marassi FM, and Opella SJ (1996) Applications of multidimensional solid-state NMR spectroscopy to membrane proteins. In: Jardetzky O and Lefevre J (eds.) Dynamics and the Problem of Recognition in Biological Macromolecules, pp. 237--255. New York: Plenum. Reid DG (ed.) (1997) Protein NMR techniques In: Methods in Molecular Biology. Totawa, New Jersey: Humana Press. Sanders CR II and Landis GC (1995) Reconstitution of membrane proteins into lipid-rich bilayered mixed micelles for NMR studies. Biochemistry 34: 4030--4040. Smith SO, Ascheim K, and Groesbeck M (1996) Magic angle spinning NMR spectroscopy of membrane proteins. Quarterly Review of Biophysics 29: 395--449. Vold RR, Prosser RS, and Deese AJ (1997) Isotropic solutions of phospholipid bicelles: A new membrane mimetic for high-resolution NMR studies of polypeptides. Journal of Biomolecular NMR 9: 329--335. Watts A, Ulrich AS, and Middleton DA (1995) Membrane protein structure: The contribution and potential of novel solid state NMR approaches. Molecular Membrane Biology 12: 233--246. Watts A (1993) Magnetic resonance studies of phospholipid–protein interactions in bilayers. In: Cevc G (ed.) Phospholipids Handbook, pp. 687--740. New York: Marcel Dekker. Watts A (1998) Solid state NMR approaches for studying the interaction of peptides and proteins with membranes. Biochimica et Biophysica Acta 1376: 297--318. Watts A (1999) NMR of drugs and ligands bound to membrane receptors. Current Opinion in Biotechnology 10: 48--53.