1.1 Biological membranes

Membranes define the physical boundaries of organelles, cells, unicellular organisms, and some viruses. Under a microscope, cell membranes appear to be continuous round or oval containers, which encase their contents, separating them from the external surroundings while providing a mechanism for selective passage of chemicals and signals between the external and internal environments. It is well known that spherical artificial membranes, also known as liposomes, can form spontaneously from phospholipids in water. Although liposomes appear to be superficially similar to biological membranes, membranes extracted from living organisms consist of ~50% protein and 50% lipid by weight. Although compartmentalization can be handled by the phospholipids alone, one-third of the proteins expressed from a typical genome are associated with membranes in order to handle the transport and signaling activities. Understanding membrane proteins demands a structural approach to characterize the factors that influence the atomic resolution structures and dynamics of the proteins and their functions within the phospholipid bilayer environment in which they reside. Because of the liquid crystalline nature of the phospholipid bilayer environment, much of this information is available only from nuclear magnetic resonance (NMR) spectroscopy. This makes studies of membranes among the most meaningful applications of NMR to structural biology.

Structure determination of membrane proteins in general has been hampered by technical difficulties stemming primarily from the preparation of samples suitable for the most widely used methods of structure determination, such as X-ray crystallography and solution NMR spectroscopy. Compared to the more familiar globular proteins, which are generally soluble and crystallizable, membrane proteins are hydrophobic, insoluble in aqueous solution, and difficult to refold into their stable, active conformation. After many years of development, solid-state NMR has matured into an approach fully capable of determining the structures of membrane proteins in their native phospholipid bilayer environment under physiological conditions, and at the present time is the only method with this capability. The initial structures of membrane proteins obtained under near-native conditions are providing a basic understanding of their structures, dynamics, and functions in biological membranes. Along the way, many studies have been performed under a wide variety of sample conditions, the best that could be done at the time, and they have contributed to the development of the spectroscopic methods and have provided background on many issues surrounding the structures and dynamics of these proteins. However, these results have to be interpreted with caution because it is known that non-native environments, such as organic solvents and detergents, can affect the structures and dynamics of membrane proteins.

The characterization of membrane proteins is built on the foundation provided by two of the earliest biophysical chemists, Christian Anfinsen and Charles Tanford. A few of their key ideas are briefly summarized here to provide context for the subsequent applications of NMR spectroscopy to membrane proteins in phospholipid bilayers, which involves the use of many additional layers of technology. Anfinsen (Reference Anfinsen1973) noted that ‘the thermodynamic hypothesis states that the three-dimensional (3D) structure of a native protein in its normal physiological milieu (solvent, pH, ionic strength, presence of other components, such as metal ions or prosthetic groups, temperature, and others) is the one in which the Gibbs free energy of the whole system is lowest; that is, that the native conformation is determined by the totality of interatomic interactions and hence by the amino acid sequence, in a given environment. In terms of natural selection through the design of macromolecules during evolution, this idea emphasized the fact that a protein molecule only makes stable, structural sense when it exists under conditions similar to those for which is was selected – the so-called physiological state.’

Amphipathic molecules, such as phospholipids, form the seamless boundaries of cells and organelles, and the associated proteins control the influx and efflux of metabolites and other biomolecules. This is consistent with the hydrophobic effect, as noted by Tanford (Reference Tanford1978), according to whom ‘the thermodynamics of biological organization does not involve biosynthesis or other chemical transformations, but focuses solely on where molecules prefer to go after they have been synthesized.’

Consistent with these principles, proteoliposomes, protein-containing phospholipid bilayers, are well-characterized and experimentally accessible versions of biological membranes. Although by necessity organic solvents and detergents were used to solubilize membrane proteins for the earliest studies, they violate these tenets, and now that solid-state NMR is providing structures under near-native conditions, many of the structures are found to differ in the non-native environments.

1.2 Biogenesis of membranes

Most discussions about the inception of life following the formation of Earth about 4·5 billion years ago are focused on molecules that evolved into the polymers that store information and carry out chemical processes. However, the emergence of membrane-like barriers to separate these molecules from the surrounding environment was a prerequisite for the formation of self-replicating systems. Membrane-like barriers must have been formed by pre-biotic amphiphiles. Notably, single-cell prokaryotes left fossil evidence dating back 3·4 billion years (Wacey et al. Reference Wacey, Kilbrun, Saunders, Cliff and Brasier2011). The amphipathic molecules that formed primitive membranes in early living systems served to maintain the proximity of a growing number of interacting functional and information-bearing macromolecules in addition to controlling the influx and efflux of metabolites.

The protein-containing membranes of contemporary organisms did not appear fully formed. Like all other aspects of biology, they are the result of billions of years of evolution and are finely tuned for their specialized biological roles. Consequently, abiogenesis includes amphiphiles for membranes which resulted, along with amino acids, nucleic acids, and carbohydrates and their precursors, from the conditions on the early Earth, as mimicked by the classic Miller–Urey experiment (Miller, Reference Miller1953).

The pre-biotic amphiphiles that assembled to form a barrier essential for the start of evolution (Lombard et al. Reference Lombard, Lopez-Garcia and Moreira2012) may bear some resemblance to the amphiphilic compounds used for solubilization and crystallization of membrane proteins. Subsequent co-evolution of proteins and phospholipids resulted in contemporary biological membranes with their unique structural and functional characteristics. Our focus in this review is on structure determination of the proteins that are associated with membranes. For the purpose of structure determination, pre-biotic amphiphiles are a starting point in the search for detergents capable of solubilizing membrane proteins, providing a chemical connection between the earliest membranes and those in mammals. In general, sample preparation and spectroscopic studies of bacterial and human membrane proteins are performed using essentially the same experimental protocols.

1.3 Phospholipid structure

Phospholipids (Cao et al. Reference Cao, Burke and Dennis2013) and proteins are the two principal macromolecular constituents of biological membranes. Some membranes also contain large amounts of cholesterol. The phospholipids found in biological membranes consist of an amphipathic or charged polar head group attached to two long hydrocarbon chains through a tri-substituted glycerol backbone. A wide range of head groups, chain lengths, number of double bonds, and other chemical characteristics are found in biological membranes. In water, phospholipids have negligible concentrations due to their low solubility, and instead, self-assemble to form bilayers, with the polar head groups exposed to water and the long hydrocarbon chains located in the hydrophobic bilayer interior shielded from the polar, aqueous environment. On the molecular scale, these bilayer sheets are infinitely long in two dimensions, but only two molecules thick in the third dimension, which is sufficient to provide a chemical and physical barrier that is so impermeable that it requires proteins to enable ions, small molecules, biopolymers, and signals to cross. Intra- and inter-molecular motions of the phospholipids add to the complexity of the membrane. In addition to their definitive gel to liquid crystalline phase transitions, different lipids have different amplitudes of motion from the surface to the center of the bilayer (Hubbell & McConnell, Reference Hubbell and McConnell1971).

Both the phospholipids and the proteins undergo fast rotational diffusion about the bilayer normal as well as translational diffusion in the plane of the bilayer and occasionally ‘flip-flop’ motions across the bilayer (Cherry, Reference Cherry1978). These global motions, which are clearly integral to the functions of membranes, cannot be mimicked in proteins solubilized in organic solvents, detergents, or frozen at low temperatures, again emphasizing the importance of studying membrane proteins as part of the membrane assembly under physiological conditions.

The basic understanding of the properties of biological membranes began to emerge in the early 1970s (Singer & Nicholson, Reference Singer and Nicholson1972). This is in contrast to the dramatic announcements that signaled the birth of molecular biology 15–20 years earlier with the structure of the DNA double helix in 1953 (Watson & Crick, Reference Watson and Crick1953) and that of the protein myoglobin in 1958 (Kendrew et al. Reference Kendrew, Bodo, Dintzis, Parrish, Wyckoff and Philips1958). All three of these classes of molecules, DNA, proteins, and lipids, as well as carbohydrates, and their partners and complexes must have evolved in concert, and are inextricably linked. As a result, the underlying theme of this review is how the liquid crystalline environment of phospholipid bilayers affects the properties of membrane proteins. This is the situation even when the membrane is reduced to its minimal chemical composition of a single type of protein-containing phospholipid that self-assembles as described by the fluid mosaic model (Singer & Nicholson, Reference Singer and Nicholson1972).

1.4 NMR of phospholipid bilayers

NMR played a key role in sorting out the properties of the various lipid assemblies studied in the early 1970s, including their implications for the structures of membrane proteins. In large part this is due to NMR being well suited for the characterization of local and global dynamics. Even when quantitative interpretations may be elusive, there is no mistaking the effects of large-amplitude, rapid motions on NMR spectra because of the dramatic narrowing of resonances that occurs.

The earliest NMR experiment on membranes focused on bilayers of pure phospholipids as well as of mixtures of lecithins. Isotopic labeling was rarely used because of its limited availability as well as the technical limitations of the commercial spectrometers. Thus, at the time, the only viable spectroscopic approach was high-resolution ¹H solution NMR, which was performed using instruments and methods developed for liquids. This limitation in spectroscopic technology contributed to their being a fundamental controversy in the field from the very beginning.

The facts were indisputable. Phospholipids in water formed bilayers that yielded very broad ¹H resonances (Chan et al. Reference Chan, Feigenson and Seiter1971, Reference Chan, Seiter and Feigenson1972, Reference Chan, Sheetz, Seiter, Feigenson, Hsu, Lau and Yau1973; Finer et al. Reference Finer, Flook and Hauser1972; Keough et al. Reference Keough, Oldfield and Chapman1973; Lichtenberg et al. Reference Lichtenberg, Petersen, Girardet, Kainosho, Kroon, Seiter, Feigenson and Chan1975; Veksli et al. Reference Veksli, Salsbury and Chapman1969). When these same samples were subjected to sonication, forming small, discrete ‘sonicated vesicles’, the spectra contained much narrower ¹H resonances. At the time, there were several theoretical interpretations to explain the drastic differences in signal properties from the same molecules in liposomes that appeared to differ only in diameter (Seiter & Chan, Reference Seiter and Chan1973). One explanation was that the lipids have basically the same properties in liposomes and vesicles, and the line narrowing resulted from the more rapid isotropic reorientation of vesicles, which could have a diameter as small as 250 Å, compared to the extended two-dimensional (2D) arrays in multilamellar liposomes. The other explanation was that the phospholipid environment in small vesicles was perturbed by the effects of sonication and the formation of a highly curved assembly, and that the resulting local disorder led to local motions of the lipid chains and line narrowing of the ¹H resonances (Sheetz & Chan, Reference Sheetz and Chan1972).

Through many studies, the properties of liposomes were characterized and it was understood that the large assemblies of lipids were ill suited for solution NMR experimental methods and theories. The major advances awaited the advent of solid-state NMR spectroscopy, which utilized ²H, ³¹P, and ¹³C signals from the phospholipids. The initial studies were from the group of Chapman, which broadly explored the application of NMR to liposomes (Keough et al. Reference Keough, Oldfield and Chapman1973; Oldfield et al. Reference Oldfield, Chapman and Derbyshire1971; Veksli et al. Reference Veksli, Salsbury and Chapman1969). There were two notable features about these studies. One was the use of a spin S=1 nucleus, deuterium, with its quadrupolar interaction available for analysis. The other was one of the first uses of isotopic labeling in biological NMR to place the deuterium nuclei at informative locations in the lipids. This addressed the limitations of using ¹H NMR to study liquid crystalline systems with extended order. In highly anisotropic environments, such as liquid crystals and lipid bilayers, each ²H nucleus gives a doublet in the spectrum. For a bilayer membrane oriented on glass plates with the bilayer normal parallel to the magnetic field, the observed splitting measures the angle between the bond and the bilayer normal (Oldfield et al. Reference Oldfield, Chapman and Derbyshire1971). In powder samples, the magnitude of the quadrupolar coupling can also be measured, as well as any reduction from the static value interpreted in terms of dynamics, and this was particularly important in making the transition from the lipid to the protein components of membranes (Blume et al. Reference Blume, Wittebort, Das Gupta and Griffin1982; Keniry et al. Reference Keniry, Gutowsky and Oldfield1984; Kinsey et al. Reference Kinsey, Kintanar and Oldfield1981a, Reference Kinsey, Kintanar, Tsai, Smith, Janes and Oldfieldb; Seelig et al. Reference Seelig, Axel and Limacher1973; Smith & Oldfield, Reference Smith and Oldfield1984).

In 1973, Urbina and Waugh described the first application of high resolution solid-state NMR to membranes. They obtained natural abundance ¹³C NMR spectra of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers above and below the phase transition. Presciently, the spectra showed more signal intensity at the lower temperature where the dipolar couplings are stronger, enabling more efficient cross-polarization from the abundant ¹H nuclei to the ‘dilute’ ¹³C nuclei. The implementation of ¹³C detection opened up many more sites, as well as the ¹³C chemical shift anisotropy and heteronuclear ¹³C–¹H dipole–dipole spin-interactions for investigation. Among the first findings about a protein embedded in a membrane bilayer was the dramatic effects of the rotational diffusion, as anticipated in the fluid mosaic model of membrane proteins, by the averaging of the ¹³C′ powder pattern in labeled bacteriorhodopsin in membrane bilayers (Lewis et al. Reference Lewis, Harbison, Herzfeld and Griffin1985).

1.5 Protein structure

The precisely folded 3D structure of a protein arranges the chemical functional groups of backbone and side chain sites so that they can carry out the necessary functions with high selectivity and efficiency, for example, catalysis of chemical reactions, transport of ions and organic molecules, binding of signaling molecules, etc. (Petsko & Ringe, Reference Petsko and Ringe2008). Generally, any variation from the native structure results in an inactive protein with accompanying consequences for biological function. The secondary and tertiary structures of a protein are commonly represented graphically by backbone ribbons (Richardson, Reference Richardson1981). These and other representations of protein structures are typically derived from many individual distances and angles between proximate atoms, as measured in X-ray diffraction or solution NMR experiments. Magic angle spinning (MAS) solid-state NMR provides similar local structural parameters. In contrast, oriented sample (OS) solid-state NMR provides primarily angular constraints measured relative to a single external axis, such as the magnetic field. In all cases, the quality of the structures benefits from the complementary measurements from the various techniques, with the primary issue being that the samples provide comparable membrane environments. Notably, the backbone structure of a protein is equivalently represented by the dihedral angles between the peptide planes in a Ramachandran plot (Ramachandran et al. Reference Ramachandran, Ramakrishnan and Sasisekharan1963).

Since protein structure determination started with X-ray diffraction of single crystals (Dickerson et al. Reference Dickerson, Reddy, Pinkerton and Steinrauf1962; Kendrew et al. Reference Kendrew, Bodo, Dintzis, Parrish, Wyckoff and Philips1958; Perutz et al. Reference Perutz, Rossman, Cullis, Muirhead and Georg1960), and the majority of new structures continue to be determined with this approach, globular proteins are often envisaged as very complex, folded polypeptides, even though they reside and function in aqueous solution where they undergo rapid isotropic reorientation. In addition, large-amplitude side chain motions are superimposed on the backbone structure of the proteins (Gall et al. Reference Gall, Cross, Diverdi and Opella1982; Wuthrich & Wagner, Reference Wuthrich and Wagner1975). Here we discuss the structure determination of membrane proteins in their native environment of phospholipid bilayers by solid-state NMR spectroscopy (Opella, Reference Opella2013b; Zhou & Cross, Reference Zhou and Cross2013), and the effects of their sequence and environment on their 3D structure (Franks et al. Reference Franks, Linden, Kunert, Van Rossum and Oschkinat2012).

Although equally true for all proteins, the combined influence on protein structure from both its sequence of amino acids and its environment is most evident in membrane proteins largely because they are embedded in highly asymmetric, liquid crystalline phospholipids, rather than an isotropic solution or a regular lattice. The nature of the membrane environment used in experimental studies, especially structure determination by NMR, of membrane proteins has been a major factor since the beginning of the field (Bosch et al. Reference Bosch, Brown and Wuthrich1980; Cross & Opella, Reference Cross and Opella1979, Reference Cross and Opella1980; Lauterwein et al. Reference Lauterwein, Bosch, Brown and Wuthrich1979). Environments ranging from mixed organic solvents to protein-containing phospholipid bilayers (proteoliposomes) (Banerjee & Datta, Reference Banerjee and Datta1983) have been used in structural and other physical and chemical studies of membrane proteins. Because of the difficulty in dealing with these highly hydrophobic proteins, often the goal of the synthetic membrane environment was simply to solubilize the polypeptides so that measurements could be made. Now, with a growing database of membrane protein structures, it is becoming apparent that detergents and organic solvents are generally problematic. The amino acid sequence alone is not sufficient to determine the 3D structure of a protein. Just as globular proteins require an aqueous environment to fold properly, membrane proteins require liquid crystalline phospholipid bilayers. Both classes of proteins require physiological conditions of temperature, pH, concentration, etc. Solid-state NMR is essential for studies of membrane proteins associated with lipid bilayers because they are effectively ‘immobilized’ by their interactions in this large supramolecular membrane structure. A variety of solid-state NMR approaches have been applied to membrane proteins and recent reviews are available on specific examples and aspects of the method (http://www.drorlist.com/nmr/MPNMR.html; Cegelski, Reference Cegelski2013; Comellas & Rienstra, Reference Comellas and Rienstra2013; Franks et al. Reference Franks, Linden, Kunert, Van Rossum and Oschkinat2012; Goncalves et al. Reference Goncalves, Eilers, South, Opefi, Laissue, Reeves and Smith2013; Hong et al. Reference Hong, Zhang and Hu2012; Judge & Watts, Reference Judge and Watts2011; Knight et al. Reference Knight, Felli, Pierattelli, Emsley and Pintacuda2013; McDermott, Reference Mcdermott2009; Mueller & Dunn, Reference Mueller and Dunn2013; Murray et al. Reference Murray, Das and Cross2013; Opella, Reference Opella2013a, Reference Opellab; Parthasarathy et al. Reference Parthasarathy, Nishiyama and Ishii2013; Popot, Reference Popot2010; Saito et al. Reference Saito, Ando and Ramamoorthy2010; Sengupta et al. Reference Sengupta, Nadaud and Jaroniec2013; Shi & Ladizhansky, Reference Shi and Ladizhansky2012; Tang et al. Reference Tang, Comellas and Rienstra2013a; Ullrich & Glaubitz, Reference Ullrich and Glaubitz2013; Ulrich, Reference Ulrich2005; Weingarth & Baldus, Reference Weingarth and Baldus2013; Yan et al. Reference Yan, Suiter, Hou, Zhang and Polenova2013).

Proteoliposomes are nearly ideal samples for structure determination of membrane proteins, since the proteins are embedded in chemically defined, fully hydrated, liquid crystalline phospholipid bilayers. This is illustrated in the iconic representation by Singer and Nicholson of the ‘fluid mosaic’ model of a biological membrane (Singer & Nicholson, Reference Singer and Nicholson1972). It is important that the forces exerted by the phospholipids on the polypeptides in proteoliposomes are likely to be very similar to those encountered in a biological membrane. Significantly, the proteins in membranes are highly constrained in most directions while undergoing two types of large-amplitude, rapid, global motions. These are fast rotational diffusion about the bilayer normal and lateral diffusion in the plane of the bilayer.

Most of the previous characterizations of membrane protein dynamics have focused on local backbone motions and those of the side chains, many of which have important implications for the functions of the proteins (Gall et al. Reference Gall, Cross, Diverdi and Opella1982; Herzfeld et al. Reference Herzfeld, Mulliken, Siminovitch and Griffin1987; Keniry et al. Reference Keniry, Gutowsky and Oldfield1984; Kinsey et al. Reference Kinsey, Kintanar and Oldfield1981a; Leo et al. Reference Leo, Colnago, Valentine and Opella1987; Wuthrich & Wagner, Reference Wuthrich and Wagner1975). Here, we focus on the influence of global rotational diffusion of the membrane proteins about the bilayer normal, not only to understand its effects on the protein structures, dynamics, and functions, but also in order to exploit this fundamental property of membrane proteins to expand the repertoire of solid-state NMR spectroscopy for protein structure determination (Das et al. Reference Das, Nothnagel, Lu, Son, Tian, Marassi and Opella2012; Lewis et al. Reference Lewis, Harbison, Herzfeld and Griffin1985; Park et al. Reference Park, Casagrande, Das, Albrecht, Chu and Opella2011b).

1.6 Global motions of membrane proteins

In aqueous solution, globular, soluble proteins undergo rapid isotropic reorientation; the rate or frequency of the motion is generally described in terms of the rotational correlation time, which is typically only somewhat slower than the timescale of a liquid, in the range of 1–20 nsec. The diameter of the protein and viscosity of the solution determine the rotational correlation time. In cases where the protein structure is non-spherical, the reorientation is found to be anisotropic. The overall rotational motions of soluble, globular proteins are well characterized by NMR and other biophysical experiments (Bauer et al. Reference Bauer, Opella, Nelson and Pecora1975) and in most cases, it is readily possible to distinguish any local backbone motions from the overall reorientation of the protein based on their differences in frequencies, amplitudes, and directions, which generally result in a sub-set of narrower, more intense, resonances superimposed on the background of broad resonances associated with the folded, structured bulk of the protein (Bogusky et al. Reference Bogusky, Schiksnis, Leo and Opella1987).

In contrast, the extremely asymmetric chemical and physical environment of phospholipid bilayers drastically affects the global motions of membrane proteins (Edidin, Reference Edidin1974). There are three major classes of motions of the protein in the bilayer to consider, two of which the NMR experiment is insensitive to. First, there are ‘flip-flop’ motions where the molecule goes from one side of the bilayer to the other. NMR is insensitive to these symmetric inversions, and flip-flop motions have generally been characterized as infrequent events, too slow to be detected by the vast majority of NMR experiments. The second is lateral diffusion in the plane of the bilayer, which has been thoroughly studied in a number of cases and may be of great importance to biological functions (Devaux & McConnell, Reference Devaux and McConnell1973; Devaux & McDonnell, Reference Devaux and Mcdonnell1972; Poo & Cone, Reference Poo and Cone1973, Reference Poo and Cone1974). However, NMR is also insensitive to translational motion.

The third type of global motion, rotational diffusion, is crucial for both the functions of the proteins and an approach to the determination of the structures in the bilayer environment. Well-understood physical laws (Durr et al. Reference Durr, Waskell and Ramamoorthy2007), two of which are directly pertinent to determining structure from global motions, govern NMR spectroscopy. One is the timescale. The frequency breadth of the spin-interactions, represented in the spectra as powder patterns, defines a ‘ruler’ for the rate at which motions affect the spectra. Motions that are ‘fast’ compared to the frequency breadth average the powder patterns, and those that are ‘slow’ do not affect the spectra. The second is the angular dependence, which has an equally strong, but entirely different effect on the spectra.

1.7 Effects of motional averaging in solid-state NMR spectroscopy

There are substantial molecular motions in seemingly rigid solids that strongly affect NMR spectra. This is not a generalized narrowing, but rather well-defined motions about a single axis that produced predictable and quantitative changes in the powder patterns that could be directly related to the line shapes observed in the same samples without motions (Gutowsky & Pake, Reference Gutowsky and Pake1950), for example at very low temperatures. Importantly, the motionally averaged powder patterns reflect the precise geometry of the internal motion about a fixed axis, and are highly relevant to the situations encountered with proteins used as samples for ¹H/¹³C/¹⁵N triple-resonance experiments. This is the key concept for deriving structural information from rotationally averaged powder patterns, which we refer to as rotationally aligned (RA) solid-state NMR. In the case of membrane proteins, we consider the vast majority of backbone N–H and C–H bonds to be rigid on the time scales of the dipole–dipole and chemical shift interactions, thus it is the motion from the global rotational diffusion of the protein in the liquid crystalline bilayer that averages the powder pattern as determined by the angle between the principal axis of the coupled pair of nuclei (Gutowsky & Pake, Reference Gutowsky and Pake1950) or the chemical shift tensor (Mehring et al. Reference Mehring, Griffin and Waugh1971) of a site and the bilayer normal (McLaughlin et al. Reference Mclaughlin, Cullis, Hemminga, Hoult, Radda, Ritchie, Seeley and Richards1975).

With the increasing interest in the analysis of the functions of membrane proteins in terms of their structures, there is a heightened concern about the influence of the surrounding environment when it is provided by detergents, non-natural lipids, organic solvents, crystal packing, etc. (Durr et al. Reference Durr, Soong and Ramamoorthy2013; Judge & Watts, Reference Judge and Watts2011; Kruger-Koplin et al. Reference Kruger-Koplin, Sorgen, Druieger-Koplin, Rivera-Torres, Cahill, Krulwich and Girvin2004; Page et al. Reference Page, Moore, Nguyen, Sharma, Chase, Gao, Mobley, Sanders, Ma, Sonnichsen, Lee, Howell, Opella and Cross2006; Poget & Girvin, Reference Poget and Girvin2007; Sanders & Oxenoid, Reference Sanders and Oxenoid2000; Vinogradova et al. Reference Vinogradova, Sonnichsen and Sanders1998). Nonetheless, considerable progress has been made in describing the structures of membrane proteins, primarily by focusing on exceptional cases, for example bacteriorhodopsin, which as a bacterial photo transducer is an extraordinarily stable protein. The early applications of electron microscopy of tilted, unstained samples to identify the seven trans-membrane TM helices of bacteriorhodopsin in its native purple membrane environment were crucial first steps in understanding the architecture of membrane proteins (Henderson & Unwin, Reference Henderson and Unwin1975). As shown by a variety of experimental measurements, bacteriorhodopsin protein molecules are constrained in their highly ordered bilayer environment containing diphytanyl lipids. However, when the protein is purified and reconstituted into bilayers consisting primarily of phosphatidyl choline lipids, it undergoes rapid rotational diffusion about the bilayer normal at temperatures above that of the gel to liquid crystalline phase transition temperature of the lipids (Lewis et al. Reference Lewis, Harbison, Herzfeld and Griffin1985). In a spectrum obtained at 3°C, well below the DMPC phase transition temperature, the chemical shift powder pattern from the carbonyl carbons has the full width and asymmetry expected in a rigid lattice. In contrast, the spectrum of the same sample obtained at 30°C, above the phase transition temperature, is much narrower and axially symmetric as the result of the motional narrowing from the rotational diffusion about the bilayer normal. This shows that the majority of labeled carbonyl groups are aligned in the protein with the C–O bond almost parallel to the bilayer normal, which is consistent with the arrangement of helices in the static structures. Similar results are obtained for other highly helical membrane proteins because the helices are aligned approximately parallel to the bilayer normal. Indeed, the ¹³C′ resonance line shape in an unoriented, stationary sample is often used as a ‘control’ experiment to ensure that the protein is undergoing rapid rotational diffusion about the bilayer normal under the experimental conditions for the structure determination.

Taken together, these early structural and dynamic studies of bacteriorhodopsin demonstrated that fast rotational diffusion of membrane proteins about the bilayer normal depends on lipid composition and temperature. Typically, the rate of rotational diffusion about the bilayer normal can be switched between frequencies that are ‘slow’ or ‘fast’ on the relevant NMR timescales defined by the frequency breadth of the static powder patterns by changing the temperature between values that are below or above, respectively, the gel to liquid crystalline phase transition temperature of the phospholipids. These experiments serve to map out the basic parameters of lipid and protein motions in artificial and biological membrane bilayers. Of particular importance to structure determination is the finding that under most conditions even quite large membrane proteins undergo fast rotational diffusion about the bilayer normal that is highly relevant to NMR spectroscopy (Park et al. Reference Park, Casagrande, Das, Albrecht, Chu and Opella2011b).

1.8 Equivalence of rotational diffusion and sample alignment

Early studies indicated that the membrane was in a ‘fluid’ state, a term that was often used synonymously with liquid crystalline state (McConnell & Hubbell, Reference McConnell and Hubbell1971; McConnell et al. Reference McConnell, Wright and Mcfarland1972; Wien et al. Reference Wien, Morrisett and Mcconnelll1972). Certainly some of the magnetic resonance experiments on synthetic and biological bilayers gave results compatible with large amplitude effectively isotropic motions because of the extensive line narrowing. However, as the research moved toward unperturbed bilayers, the asymmetric nature of phospholipid bilayers dominated the effects. As mentioned above, early critical experiments utilized rhodopsin, especially those of Cone (Cone, Reference Cone1972), which measured the relaxation due to rotation about an axis normal to the plane of the disc membrane.

The rotational diffusion of membrane proteins merges OS solid-state NMR and MAS solid-state NMR experimental methods enabling the complementary use of aligned and unoriented samples, and increasing the potential for synergy between isotropic and anisotropic nuclear spin interaction contributions to protein structure determination. This is feasible because the same rotationally averaged powder patterns observed in stationary unoriented samples can be measured in MAS solid-state NMR spectra of unoriented samples through the application of ‘slow spinning’ (Herzfeld & Berger, Reference Herzfeld and Berger1980; Wylie et al. Reference Wylie, Sperling, Frericks, Shah, Franks and Rienstra2007) or recoupling methods (Chan & Tycko, Reference Chan and Tycko2003; Wylie et al. Reference Wylie, Franks and Rienstra2006). Here, we use the spectral parameters associated with the ¹⁵N and ¹³C chemical shifts and ¹H–¹⁵N and ¹H–¹³C dipolar couplings in specifically labeled backbone and side chain sites to demonstrate the equivalence of mechanical and magnetic uniaxial alignment of protein-containing bilayers and ‘rotational alignment’ of the membrane proteins resulting from their rapid rotational diffusion about the bilayer normal in unoriented samples when the temperature is higher than that of the gel to liquid crystalline transition for the phospholipids. Because the resonance frequencies are determined by the orientations of functional groups (chemical shift) or chemical bonds (dipole–dipole coupling) relative to a single axis defined by the direction of the magnetic field or by rotational diffusion about the bilayer normal, the protein structure is mapped onto the resulting spectra by the anisotropy of the spin interactions.

These measurements are well suited for membrane proteins that undergo rapid rotational diffusion in phospholipid bilayers. Also, these measurements are particularly forgiving with respect to experimental errors and uncertainties regarding the principal values and orientations of the tensors in the molecular frame because site-to-site errors do not accumulate. Each measurement is made independently relative to a common external axis, in this case the bilayer normal. In complementary approaches, the axis can be in the direction of the magnetic field, orthogonal to the field, or relative to the mechanical alignment on glass plates (Park et al. Reference Park, Das, Deangelis, Scrima and Opella2010a).

Uniaxial sample alignment, where the direction of the molecular alignment is parallel to that of the magnetic field, is the next most general type of sample after a single crystal. In this case, the sample has one direction of orientation and the other two directions are random or undefined. What is most notable is that while these samples are capable of yielding very high-resolution spectra when they are aligned parallel to the field, the line shapes are 2D powder patterns when they are tilted away from parallel (Opella & Waugh, Reference Opella and Waugh1977). This is seen most dramatically when the axis of alignment is perpendicular to the direction of the field. However, the situation is greatly simplified when the molecule of interest undergoes fast rotational diffusion about a single axis, in which case single line spectra are observed in all directions of alignment (Park et al. Reference Park, Mrse, Nevzorov, De Angelis and Opella2005).

Membrane proteins in phospholipid bilayers can be aligned mechanically between glass plates (Bechinger et al. Reference Bechinger, Kim, Chirlian, Gesell, Neumann, Montal, Tomich, Zasloff and Opella1991; Murray et al. Reference Murray, Das and Cross2013). As with uniaxially aligned polymers, when the axis of alignment is parallel to the field single line resonances are observed regardless of whether any molecular motion is present. Magnetically aligned samples of membrane proteins were originally sought as direct replacements for mechanically aligned samples (De Angelis et al. Reference De Angelis, Nevzorov, Park, Howell, Mrse and Opella2004) and once it was discovered that the lanthanide ions added to the lipids could ‘flip’ the alignment so that the bilayer normals were parallel to the field (Prosser et al. Reference Prosser, Hunt, Dinatale and Vold1996), then that was the case. However, the presence of detergents to form the ends of the bilayer discs and lanthanides is of concern for their possible effects on the protein structures of interest.