[19] Experimental nuclear magnetic resonance studies of membrane proteins

536

PROTEIN STRUCTURE

[19]

[19] E x p e r i m e n t a l N u c l e a r M a g n e t i c R e s o n a n c e S t u d i e s o f Membrane Proteins

By S. J. OPELLA,Y.

KIM,

and P. MCDONNELL

Introduction

Understanding the biological properties and functions of proteins associated with membranes requires the same kind of high-resolution structural analysis that has been performed on many soluble proteins of the cytoplasm and periplasm. In general, however, this has not been feasible, largely because samples of membrane proteins are problematic for the most commonly used methods of structural biology. Membrane-associated proteins are difficult to crystallize in forms suitable for X-ray diffraction, and the multidimensional solution nuclear magnetic resonance (NMR) methods described in detail in other chapters of this volume are hampered by the slow overall reorientation rates of proteins complexed with lipids. An important goal of structural biology is to develop methods capable of determining the structures of membrane proteins, since they are responsible for a variety of unique functions in biological systems, many of which have direct consequences for medicine and biotechnology. NMR spectroscopy will play a prominent role in the characterization of membrane proteins; not only is it versatile enough to determine the structures of proteins in partially ordered complexes with lipids, it lends itself to describing intramolecular motions. This chapter presents the major features of an experimental NMR approach for describing membrane proteins based on combining the results of solid-state NMR experiments on oriented and unoriented samples of proteins in hydrated phospholipid bilayers with those from multidimensional solution NMR experiments on samples of proteins in detergent micelles in aqueous solution. Although at an early stage in its development, this approach has been successfully applied to several membrane-associated peptides 1'2 and proteins. 3-7 t B. Bechinger, Y. Kim, L. E. Chirlian, J. Gesell, J. M. Neumann, M. Montal, J. Tomich, M. Zasloff, and S. J. Opella, J. Biomol. N M R 1, 167 (1991). -~S. J. Opella, J. Gesell, and B. Bechinger, in "The Amphipathic Helix" (R. Epand, ed.), p. 87. CRC Press, Boca Raton, Florida, 1993. 3 K. J. Shon, Y. Kim, L. A. Colnago, and S. J. Opella, Science 252, 1303 (1991). 4 K. J. Shon, P. Schrader, Y. Kim, B. Bechinger, M. Zasloff, and S. J. Opella, in -Biotechnology: Bridging Research and Applications" (D. Kamely, A. Chakrabarty, and S. Kornguth, eds.), p. 109. Kluwer Academic Publishers, Dodrecht, The Netherlands, 1991. 5 p. A. McDonnell, K. J. Shon, Y. Kim, and S. J. Opella, J. Mol. Biol. 233, 447 (1993).

METHODS IN ENZYMOLOGY, VOL. 239

Copyright © 1994 by Academic Press, lnc, All rights of reproduction in any form reserved.

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In spite of the formidable technical difficulties encountered in both crystallographic and spectroscopic studies of membrane proteins, the structures of several membrane proteins have been determined at relatively high resolution. 3'5'8-~1 The structures of these proteins have the important role of defining the basic architecture of membrane proteins; this establishes a basis for the design and evaluation of methods capable of determing the structures of other examples. Proteins with hydrophobic or amphipathic transmembrane and amphipathic bridging helices in dominant organizational and structural roles are of particular interest because they are responsible for many membrane-associated functions. Substantial progress can be made in determining their structures by describing, first, the dynamics and secondary structure of individual residues and, then, the orientations of segments of secondary structure relative to the lipid bilayers, all of which can be accomplished with NMR experiments. Phospholipid bilayers and detergent micelles are two well-characterized model membrane systems available for biophysical studies 12,1~ that enable both solid-state NMR and multidimensional solution NMR methods to be applied to membrane proteins. Additional development is required before these methods can be used individually to determine the structures of membrane proteins. However, results from these two essentially independent spectroscopic approaches can be combined to describe features of membrane proteins that would otherwise be inaccessible to any single method of structure determination at the present time. The combination of solid-state NMR spectroscopy of oriented and unoriented bilayer samples and multidimensional solution NMR spectroscopy of micelle samples is most effective when the 6 S. J. Opella and P. A. McDonnell, in "NMR of Proteins" (A. M. Gronenborn and G. M. Clore, eds.), p. 159. Macmillan, New York, 1993. 7 S. J. Opella, in "'Membrane Protein Structure: Experimental Approaches" (S. H. White, ed.), in press. Oxford Univ. Press, Oxford, 1994. J. Deisenhofer, O. Epp, K. Miki, R. Huber. and H. Michel. Nature (London) 318, 618 (1985). 9 D. Rees. H. Komiga, T. Yeates, J. Allen, and G. Feher, Annu. Rev. Biochem. 58, 607 (1989). ~0 R. Henderson, J. Baldwin, T. Cesko, F. Zemlin, E. Beckmann, and K. Downing, J. Mol. Biol. 213, 899 (1990). ~ M. Weiss, U. Abele, J. Weckesser, W. Welte, E. Schiltz, and G. Schulz, Science 254, 1627 (1991). i_, D. M. Small (ed.), "Handbook of Lipid Research 4: The Physical Chemistry of Lipids." Plenum, New York, 1986. i~ H. Michel (ed.), "Crystallization of Membrane Proteins." CRC Press, Boca Raton. Florida, 1991.

538

PROTEIN STRUCTURE

A

[19]

B

FIG. 1. Representations of model membranes. (A) Micelle; (B) bilayer.

proteins are suitably labeled with stable isotopes and the samples are carefully prepared. In this approach, the secondary structure of the protein is determined on the basis of internuclear distance measurements in micelle samples, and the arrangement of the major elements of secondary structure is derived from measurements of angular parameters in oriented bilayer samples. The dynamics of backbone and side-chain sites are described over a wide range of time scales through the analysis of nuclear spin relaxation in both micelle and bilayer samples, as well as motionally averaged powder-pattern line shapes in bilayer samples. 14-16 Local protein motions have a dramatic effect on many spectral parameters; therefore, mobile residues in loop and terminal segments can be readily identified in both types of samples. 17 Figure 1 presents schematic diagrams of the two different types of model membrane samples. Each micelle contains approximately the same number of detergent molecules (50-60 for the most commonly used detergents) and a single polypeptide chain. 18a9 In contrast, lipid bilayers are supramolecular structures with many phospholipid and polypeptide molecules in extended two-dimensional arrays. The molecular arrangements shown in Fig. ! strongly influence the design and implementation of the NMR experiments; proteins in micelles reorient rapidly enough to be suitable for multidimensional solution NMR experiments, whereas those in bilayers are immobile on NMR time scales, enabling them to be treated spectroscopically as solids. 14 D. A. Torchia, Annu. Rev. Biophys. Bioeng. 13, 124 (1984). I5 E. Oldfield, R. A. Kinsey, and A. Kintanar, this series, Vol. 88, p. 310. ~6 S. J. Opella, this series, Vol. 131, p. 327. i7 M. J. Bogusky, R. A. Schiksnis, G. C. Leo, and S. J. Opella, J. Magn. Reson. 72, 186 (1987). i8 j. Lauterwein, C. Bosch, L. Brown, and K. W~thrich, Biochim. Biophys. Acta 556, 244 (1979). 19 A. Helenius, D. R. McCaslin, E. Fries, and C. Tanford, this series, Vol. 56, p. 734.

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Solid-State Nuclear Magnetic Resonance Spectroscopy Proteins in Phospholipids Peptides and proteins that are strongly associated with phospholipids are well suited for solid-state NMR spectroscopy. ~5'2°-22 In most cases, the structured regions of the polypeptides are essentially completely immobilized on the time scales defined by the spectral ranges (103-106 H z ) of the chemical shift, dipolar, and quadrupolar spin interactions of the ~H, 2H, 13C, 14N, and ~SN nuclei in proteins. This immobilization preserves the anisotropic characteristics of the nuclear spin interactions averaged out by the rapid isotropic reorientation that occurs in solution, including proteins in micelles. Because it is possible to prepare both oriented and unoriented samples of proteins in fully hydrated phospholipid bilayers, a wide variety of spectral parameters can be resolved and measured from single-line and powder-pattern spectra obtained with solid-state NMR experiments. In some cases, the proteins undergo rapid axial rotation within the bilayers; this can be advantageous because the characteristically motionally averaged spectral parameters bear information about the orientations of sites in the protein 23'24 and because it allows greater choice in orienting media, especially the use of mixtures of phospholipids and detergents that orient perpendicular to the direction of the applied magnetic field. 25'26 Sample Preparation At the present time, the only reliable way to orient uniaxially peptides or proteins for NMR studies with their transmembrane helices parallel to the direction of the applied magnetic field is in hydrated phospholipid bilayers between glass plates arranged so that the bilayer normal is perpendicular to the magnetic field. ~,27-31Magnetic alignment of lipid preparations 20 R. G. Griffin, this series, Vol. 72, p. 108. 21 S. O. Smith and R. G. Griffin, Annu. Rev. Phys. Chem. 39, 511 (1988). 22 S. O. Smith and O. B. Peersen, Annu. Rev. Biophys. Biomol. Struct. 21, 25 (1992). 23 B. A. Lewis, G. S. Harbison, J. Herzfeld, and R. G. Griffin, Biochemistry 24, 4671 (1985). 24 R. S. Prosser, J. H. Davis, F. W. Dahlquist, and M. A. Lindorfer, Biochemistry 30, 4687 (1991). 25 C. R. Sanders and J. H. Prestegard, Biophys. J. 58, 447 (1990). _,6j. Seelig, F. Borle, and T. A. Cross, Biochim. Biophys. Acta 814, 195 (1985). 27 B. A. Cornell, F. Separovic, A. Baldassi, and R. Smith, Biophys. J. 53, 67 (1988). 28 R. Smith, D. Thomas, F. Separovic, A. R. Atkins, and B. A. Cornell, Biophys. J. 56, 307 (1989).

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PROTEIN STRUCTURE

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generally occurs such that transmembrane helices would be perpendicular to the field. 25'z6 It would be highly desirable to be able to use magnetic rather than mechanical orientation of the samples; this is feasible only with a few specialized preparations at the present time. 32-33 There are several ways to prepare samples of peptides and proteins in lipid bilayers for solid-state NMR experiments, depending on their source and properties. Peptides produced by automated solid-phase peptide synthesis are typically dissolved directly in an organic solvent that is also capable of dissolving the lipids of interest; some of the most commonly used solvents are 2,2,2-trifluoroethanol (TFE), 5% ethanol in benzene, 2,2,2,3,3,3-hexafluoropropanol (HFP), chloroform, methanol, and dichloromethane. For example, 40 mg of a synthetic 23-residue amphiphathic channel peptide is dissolved in less than 1 ml of TFE (Aldrich Chemical Company, Milwaukee, WI, NMR grade), and then 1,2-dimyristoyl-snglycero-3-phosphocholine (DMPC) (Sigma Chemical Company, St. Louis, MO) is added in a peptide to lipid molar ratio of 1 : 10 to prepare the sample used to obtain the spectrum of a peptide in unoriented bilayers shown in Fig. 3C. Residual insoluble material is removed by centrifugation, and the solvent is removed under vacuum. It is essential to eliminate all traces of organic solvent from the sample before rehydrating the peptide-lipid mixture by addition of distilled, deionized water. The resulting sample is a thick paste which is subjected to multiple freeze-thaw cycles in liquid nitrogen and hot water baths to ensure complete mixing of the components. Polypeptides expressed directly in bacteria or as fusion proteins are treated somewhat differently than synthetic peptides. In most cases they are transferred from detergent to lipid environments. The procedures are tailored to individual proteins; for example, filamentous bacteriophage coat protein can be directly transferred from virus particles into DMPC bilayers. 34 Phospholipids are uniformly suspended in 10 mM borate buffer at pH 8.4 along with sufficient virus for a protein to lipid molar ratio of 1:10. The mixture is sonicated until the solution becomes transparent using the low power setting (2-3) on a Branson sonifier (Model 450) with a microtip while maintaining the sample at a temperature (25°-30°), slightly

29 F. Moll and T. Cross, Biophys. J. 57, 351 (1990). 3o A. Hing, S. Adams, D. Silbert, and R. Norberg, Biochemistry 29, 4144 (1990). 3Ej. A. Killian, M. J. Taylor, and R. E. Koeppe, Biochemistry 31, 11283 (1992). 32 j. H. Davis, Biochemistry 27, 428 (1988). 33 C. R. Sanders, J. E. Schaff, and J. H. Prestegard, Biophys. J. 64, 1069 (1993). 34 S. P. Fodor, A. K. Dunker, D. Carsten, and R. W. Williams, in "Bacteriophage Assembly" (M. S. Dubow, ed.), p. 441. Alan R. Liss, New York, 1981.

[19]

N M R STUDIES OF MEMBRANE PROTEINS

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higher than the gel to liquid crystalline-phase transition temperature of the phospholipids. The sample is then lyophilized and rehydrated by addition of distilled, deionized water. Phosphorus-31 NMR spectra are useful in demonstrating that the lipids in the sample are in bilayers. The characteristic motionally averaged 31p chemical shift anisotropy powder-pattern line shape of phospholipids in the lamellar liquid crystalline phase is distinctly different from that observed for isotropic or hexagonal phases. 35 Even small amounts of lipids in nonbilayer phases resulting from the effect of the protein on the lipids or sample handling can be detected. Similarly, the single-line 31p NMR spectra from oriented bilayer samples provide important controls for both the lipid phases and sample orientation. 36,37 The choice of organic solvent for codissolving the lipids and proteins influences the preparations of oriented samples, and it is often necessary to try several different solvents to obtain optimal results. Approximately 0.1 ml of a solution containing the protein and lipids in a molar ratio of 1 : 20 is spread onto the surface of a thin glass plate (22 x 22 mm) which has been thoroughly cleaned and prewashed with chloroform and methanol. After evaporating the solvent from the material deposited on the glass plate under a stream of nitrogen gas, more of the solution containing the protein and lipids is layered on top of the dried material. This is repeated until a total of 100-150 mg of the lipid and protein mixture is uniformly distributed on one face of the plate. Residual traces of the organic solvent are removed by placing the sample under vacuum overnight at room temperature. After the sample is hydrated by the addition of 200-400/~1 of distilled, deionized water directly to the dry lipid-protein mixture on the glass plate, it is placed in a dessicator equilibrated at 93% relative humidity [with a (NH4)HzPO4-saturated salt solution] for an extended period of time, hours to weeks, depending on the sample, during which macroscopic sample orientation occurs. The sample is equilibrated at a temperature above the transition temperature of the lipids for at least 2 hr, and then a second clean glass plate is placed on top of the hydrated, oriented sample, forming a glass-bilayer-glass sandwich. Gentle finger pressure is applied symmetrically to both sides of the sandwich prior to wrapping it in Parafilm and Teflon tape. Hydration and orientation of the samples are maintained in the probe of the spectrometer by prehumidifying the air used for temperature con35 p. R. Cullis and F. B. Dekruijf, Biochim. Biophys. Acta 559, 399 (1979). 36 A. C. McLaughlin, P. R. Cullis, M. A. Hemminga, D. I. Hoult, G. K. Radda, G. A. Ritchie, P. J. Seeley, and R. E. Richards, FEBS Lett. 57, 213 (1975). 37 R. Griffin, L. Powers, and P. Persham, Biochemisoy 17, 2718 (1978).

542

PROTEIN STRUCTURE

[19]

I

FIG. 2. Flat-coil probe for solid-state NMR double-resonance experiments. 38

trol. The sample temperature is maintained above the gel to liquid crystalline phase-transition temperature of the lipids at all times. Between experiments, samples are stored in a desiccator at 93% relative humidity after loosening the wrapping to allow equilibration. Properly maintained samples retain their orientation for many months. Instrumentation

Solid-state NMR experiments on membrane protein samples are quite demanding in terms of sensitivity, requiring a high-field solid-state NMR spectrometer with high stability for long-term signal averaging. Many previous NMR studies of oriented membrane samples utilized stacks of small glass plates in order to build up a sample with sufficient material for adequate sensitivity within a solenoidal coil. However, this has substantial limitations because of variations in the orientation of the various glass plates, edge effects, and the poor filling factor of the coil. We have developed a fiat coil probe for solid-state NMR experiments on oriented bilayer samples 38in order to minimize edge effects and misalignments of stacking. By using a single pair of square glass plates with the radio frequency (rf) coil wrapped directly around them, both probe performance and sample orientation are improved. Figure 2 illustrates a fiat-coil probe, double38 B. Bechinger and S. J. Opella, J. Magn. Reson. 95, 585 (1991).

[19]

N M R STUDIES OF MEMBRANE PROTEINS

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tuned for ~H and ~SN experiments. The filling factor of the coil is near optimal which accounts for the high sensitivity and rf performance of these probes. We have constructed flat-coil double-resonance probes with ~H resonance frequencies between 150 and 550 MHz. These probes require a wide-bore magnet for adequate space and field homogeneity over the entire sample.

Dynamics The initial step in the investigation of a membrane protein is to verify that the polypeptide chain is immobilized by its interactions with phospholipids. This is accomplished most directly by observing powderpattern resonances from labeled sites in unoriented samples and comparing their shape and breadth to those from rigid polycrystalline samples. These comparisons can also provide the basis for qualitatively describing the dynamics of individual sites in the polypeptide backbone. Solid-state NMR spectroscopy can readily identify mobile residues in an immobilized protein, because powder-pattern line shapes are strongly affected by motional averaging. ~7'39 Residues that undergo large-amplitude motions more often than the frequency breadth of the powder pattern from the nuclear spin interactions of interest are particularly easy to recognize because they have narrow resonance lines at the average, isotropic frequency. The spectrum in Fig. 3A clearly demonstrates that it is possible to observe narrow isotropic resonance intensity arising from the mobile sites superimposed on the broad powder-pattern line shape from the structured sites. Resonances from all nitrogen sites in filamentous bacteriophage fd coat protein contribute to the spectrum in Fig. 3A, since it was obtained on a sample of uniformly 15N-labeled protein. The narrow peak near 15 ppm from amino groups is not particularly informative. The narrow and broad intensity between 30 and 200 ppm from the backbone amide sites is of greater interest. The mobile sites near the N and C termini and the loop connecting the helices in the protein are responsible for the relatively narrow resonance intensity superimposed on the underlying powder pattern from the majority of the backbone sites which are structured and immobile. In contrast, the spectrum in Fig. 3B consists solely of a powder pattern, because the two JSN-labeled residues, L14 and L41, are in helices immobilized by their interactions with the lipids. Likewise, the spectrum in Fig. 3C shows that the multiple labeled sites in a helical peptide are structured and immobile. 39 M. Keniry, H. Gutowsky, and E. Oldfield, Nature (London) 307, 383 (1984).

544

PROTEIN STRUCTURE

[191

A

260

6 ppm

FIc. 3. Solid-state ~SN NMR spectra of fd coat protein and M28 channel peptide in unoriented phospholipid bilayer samples. (A) Uniformly 15N-labeled fd coat proteinS; (B) selectively [15N]Leu-labeled fd coat proteinS; (C) 11 site {[~SN]Ala (6, 11, and 13), [15N]Ser (4, 8, and 20), and [JSN]Leu (9, 10, 16, 17, and 18)}-labeled M28 channel peptide; (D) simulated powder pattern for an immobile 15N amide site.

As shown in Fig. 3, 15N-labeled samples are particularly useful in describing the dynamics of peptides and proteins in bilayers17; however, because the amide 15N chemical shift tensor is nearly axially symmetric with trll approximately parallel to the N - H bond, 4°-42 it is necessary to 4o G. S. Harbison, L. W. Jelinski, R. E. Stark, D. A. Torchia, J. Herzfeld, and R. G. Griffin, J. Magn. Reson. 60, 79 (1984); T. G. Oas, C. J. Hartzell, F. W. Dahlquist, and G. P. Drobny, J. Am. Chem. Soc. 109, 5962 (1987). 41 C. J. Hartzell, M. Whitfield, T. G. Oas, and G. P. Drobny, J. Am. Chem. Soc. 109, 5966 (1987). 42 Q. Teng and T. A. Cross, J. Magn. Reson. 85, 439 (1989).

[19]

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o b s e r v e p o w d e r patterns from other sites in order to establish if the protein is undergoing rotation about an axis that is coincident with t r a n s m e m b r a n e helices within the bilayer. The 2H quadrupole p o w d e r pattern f r o m N - D (but not C - D ) sites is convenient for this purpose 43'44 since it has substantial deviations f r o m axial s y m m e t r y ; the labeling can be performed by solvent exchange, and resolution a m o n g individual sites is not required. 13C-Labeled carbonyl sites can also be used to detect rapid axial rotation of the protein in bilayers, although interference from natural abundance background means that difference s p e c t r o s c o p y m a y be needed. Some rigid side chains have sites with nonaxially symmetric chemical shift tensors that can be 13C or ~SN labeled, such as the indole nitrogen of tryptophan, 44 and they can be used to monitor the overall motion of the protein in the bilayers. Solid-state 2H N M R spectra of C - D - l a b e l e d side chains are also sensitive indicators of local motions. 14-16'39'45 The hop motions of side chains with rigid b a c k b o n e s characteristically alter powder-pattern line shapes; however, b a c k b o n e motions of substantial amplitude are required to give isotropic signals. Structure

Solid-state N M R spectroscopy is a well-established method for structure determination. Direct m e a s u r e m e n t s of both distances and orientations can be made in experiments on both oriented and unoriented samples, and several different solid-state N M R approaches to structure determination are under development, including methods based on distance measurements in unoriented samples. 46-49 The solid-state N M R a p p r o a c h to protein structure determination that we are developing 5°-52 is designed for samples that fulfill two conditions: the proteins are immobile on the appropriate N M R time scales and uniaxially oriented along the direction 43 K. Pauls, A. MacKay, O. Soderman, M. Bloom, A. Tanjea, and R. Hodges, Eur. Biophys.

J. 12, 1 (1985). 44G. C. Leo, L. A. Colnago, K. G. Valentine, and S. J. Opella, Biochemistry 26, 854 (1987). 45 D. M. Rice, A. Blume, J. Herzfeld, R. J. Wittebort, T. H. Huang, S. K, Das Gupta, and R. G. Griffin, Biomol. Stereodyn. Proc. Syrup. 2, 255 (1981). 46 D. Raleigh, M. Levitt, and R. Griffin, Chem. Phys. Lett. 146, 71 (1988). 47A. E. McDermott, F. Creuzet, R. G. Griffin, L. E. Zawadzke, Q. Z. Ye, and C. T. Walsh, Biochemistry 29, 5767 (1990). 48T. Gullion and J. Schaefer, J. Magn. Resort. 81, 196 (1989). 49G. R. Marshall, D. D. Beusen, K. Kociolek, A. S. Radlinski, M. T. Leplawy, Y. Pan, and J. Schaefer, J. Am. Chem. Soc. 112, 963 (1990). 50S. J. Opella, P. L. Stewart, and K. G. Valentine, Q. Rev. Biophys. 19, 7 (1987). 51 S. J. Opella and P. L. Stewart, this series, Vol. 176, p. 242. 52 L. E. Chirlian and S. J. Opella, Adv. Magn. Reson. 14, 183 (1990).

546

PROTEIN STRUCTURE

[19]

of the applied magnetic field. Membrane proteins are particularly well suited for this approach, since their structured regions are immobilized by interactions within the protein and with surrounding phospholipids, and protein-containing bilayers are readily oriented between glass plates. The measurement of several orientationally dependent spectral parameters for sites on each residue enables the structure of the entire protein to be determined. This solid-state NMR method relies on the spectral simplifications that result from uniaxial sample orientation parallel to the direction of the applied magnetic field. 53The observed values of the frequencies and splittings depend on the orientations of the principal axes of the spin interaction tensors present at each site relative to the direction of the applied magnetic field. Because the direction of sample orientation and the applied magnetic field of the NMR spectrometer is the same, it defines a frame of reference for the evaluation of the orientational information. The orientations of many spin interaction tensors have been established in their molecular frames of reference, enabling angular factors to be determined from the experimental data. Molecular structures can be determined on the basis of angles alone, given standard bond lengths and geometries, making it possible to determine the structure of a protein with a sufficient number of orientationally dependent spectroscopic measurements. We have demonstrated the feasibility of the method with the coat protein of filamentous bacteriophages oriented by the magnetic field of the spectrometerfl ° This approach has also been applied to magnetically oriented protein crystals 54 as well as peptides, 1'2 especially gramicidin, 26-32'55 and proteins 3-7 in oriented model membrane samples. Complete backbone structures require that several spectral parameters be measured to characterize the angles between each of the peptide planes and the direction of sample orientation. At the present time, this requires extensive isotopic labeling for resolution and assignment of the resonances from the residues. If the secondary structure of a membrane-bound form of a protein is established on the basis of homonuclear ~H nuclear Overhauser effect (NOE) and other measurements in micelle samples, a single spectral parameter, for example, the 15N r e s o n a n c e frequency in an oriented bilayer sample, is sufficient to establish the orientations of the secondary structure containing the labeled residue relative to the plane of the bilayer. For example, Fig. 4 presents solid-state 15N NMR spectra of amphipathic helical peptides in oriented bilayers, both of which have a single ~SN53 S. J. Opella and J. S. Waugh, J. Chem. Phys. 66, 4919 (1977). 54 T. Rothgeb and E. Oldfield, J. Biol. Chem. 256, 1432 (1981). 55 W. Mai, W. Hu, C. Wang, and T. A. Cross, Protein Sci. 2, 532 (1993).

[19]

N M R STUDIES OF MEMBRANE PROTEINS

O'll

547

G±

z6o

6 ppm

FIG. 4. Solid-state tSN NMR spectra of amphiphathic helical peptides in oriented lipid bilayer samples. (A) [15N]Ala-15-1abeled magainin 2 in POPC/POPG (l-palmitoyl-2-oleoylsn-glycerophosphocholine/l-palmitoyl-2-sn-glycero-3-phosphoglycerol)l; (B) [15N]Ala-12-labeled M28 channel peptides in DMPC/DMPG (1,2-dimyristoyl-sn-glycero-3-phosphocholine/ 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol). ~ The plane of the bilayers is perpendicular to the direction of the applied magnetic field.

labeled residue near the middle of the helix. Strikingly, the M28 channel peptide has its ~5N resonance separated from that of magainin by nearly the full breadth of the powder pattern for the 15N amide chemical shift interaction (see Fig. 3); this indicates that the planes containing the labeled peptide groups have very different orientations relative to the direction of the applied magnetic field. These oriented solid-state NMR spectra can be interpreted with considerable confidence, since the observed resonance frequencies are near the discontinuities of the powder pattern for the amide group, which allows the nearly axially symmetric ~SN amide chemical shift tensor 4°-42 to be used as a qualitative guide to the orientation of the peptide groups within the helices. The M26 channel peptide is transmembrane, since it is helical and the N - H bond of the labeled amide site is approximately parallel to the direction of the applied magnetic field. In contrast, the magainin peptide resides in the plane of the bilayer, since it is helical and the N - H bond of the labeled amide site is approximately perpendicular to the field. Multidimensional Solution Nuclear Magnetic Resonance Spectroscopy Proteins in Micelles

Many biophysical and biochemical studies of membrane proteins, including multidimensional solution NMR spectroscopy, are feasible only

548

PROTEIN STRUCTURE

[19]

because detergent micelles effectively mimic the molecular environment of membranes. Proteins and peptides associated with micelles undergo isotropic reorientation in solution with rotational correlation times of 20-30 × 10 -9 sec, which is slow but still suitable for experiments that utilize the instrumentation and methods of multidimensional solution NMR spectroscopy. The broad line widths and efficient spin diffusion that result from the slow reorientation of proteins in micelles combine to make homonuclear solution NMR experiments difficult. The incorporation of stable isotopes, optimization of all sample conditions, and use of high-field spectrometers are essential for multidimensional solution NMR experiments to be effective in describing the structure and dynamics of peptides and proteins in micelles. 3-5'56,57

Sample Preparation Multidimensional solution NMR methods can be applied to membrane proteins in micelles in aqueous solution and give reliable results as long as the micelle samples are carefully prepared; the choice of detergent and concentration are among the most important aspects of sample preparation. 57 Because there are no general guidelines for selecting the best detergent for a given protein or peptide, it is necessary to try out several different combinations of detergents, counterions, pH values, and temperatures in order to optimize the quality of the spectra. Sodium dodecyl sulfate (SDS) and dodecylphosphocholine (DPC) are the most commonly used detergents for NMR studies of membrane proteins in solution, because they form stable micelles with small aggregation numbers, is,J9 Both SDS and DPC are available commercially win a perdeuterated form (Cambridge Isotope Laboratories, Andover, MA), which is convenient for minimizing spectral interference from background IH resonances; however, the use of isotope-edited experiments on JSN- and/or ~3C-labeled samples, especially with gradient spectroscopy, enables the use of unlabeled detergents in these experiments? 8 The detergent used for solubilization of a protein should be of very high chemical purity, which typically requires that it be recrystallized, in some cases repeatedly. Simply solubilizing a protein with a detergent is unlikely to give samples that yield reproducible spectra, much less meaningful results. This has been illustrated for the membrane-bound form of fd coat protein, whose two-dimensional heteronuclear correlation spectra vary dramatically depending on the choice and concentation of the detergent. Samples with 56 K. Shon and S. J. Opella, J. Magn. Reson. 82, 193 (1989). 57 p. A. McDonnell and S. J. Opella, J. Magn. Reson. B, 102, 120 (1993). 58 j. Anglister, S. Grzesiek, H. Ren, C. B. Klee, and A. Bax, J. Biomol. N M R 3, 121 (1993).

[19]

N M R STUDIES OF MEMBRANE PROTEINS 8

549

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o

o

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8.0

Fro. 5. Two-dimensional IH/]SN solution NMR spectra offd coat protein in SDS micelles. 5 Heteronuclear multiple-quantum spectra of uniformly ~SN (A, B, D, E)- and selectively [tSN]lysine(C, F)-labeled coat protein in solutions with 200 mM SDS (A, B, C) and 480 mM SDS (D, E, F).

SDS concentrations 20-350 times higher than the critical micelle concentration are essential for multidimensional solution NMR studies of this protein. All amide resonances are single lines, and their widths in both the 1H and ~SN dimensions are significantly narrower at high detergent concentrations?7 The two-dimensional heteronuclear correlation spectra in Fig. 5 of uniformly and selectively []SN]lysine-labeled fd coat protein in 200 mM SDS have an apparent doubling of some amide resonances, especially those from residues in the hydrophobic membrane-spanning helix as seen in spectra obtained on samples with low detergent concentrations. 59 However, when the SDS concentration is increased to 480 mM no doubling of resonances is observed. 4.5,57 Under appropriate sample conditions, very high quality two- and threedimensional spectra that exhibit no unusual features can be obtained from membrane proteins in SDS micelles. Proteins solubilized in DPC generally do not exhibit spectral complexity, even at relatively low detergent concentrations, and many investigators are more comfortable with interpretations based on data obtained with the phosphocholine rather than sulfate 59 G. D. Henry and B. D. Sykes, Biochemist~ 31, 5284 (1992).

550

PROTEIN STRUCTURE

[19]

head group. However, in some cases solubilization of the peptide or protein in SDS rather than DPC gives narrower lines, and, as long as the sample is carefully prepared, there are no spectral complications. There are now a sufficient number of examples of peptides and proteins studied in SDS and DPC to provide assurances that both detergents provide reasonable model membrane environments. Even with optimal sample conditions, proteins and peptides in micelles reorient relatively slowly, which results in line widths larger than most homonuclear spin-spin coupling constants. This limits the applicability of many homonuclear correlation experiments, making resonance assignments challenging. In most cases, stable isotopes (~SN and/or ~3C or 2H) must be incorporated into the polypeptide, so that multidimensional heteronuclear experiments can be used for resonance assignments and structure determinations. Resonances from residues in regions of secondary structure in membrane proteins that are transmembrane generally have larger line widths and smaller homonuclear ~H NOEs to resonances from adjacent residues than those in regions of secondary structure that are in the plane of the lipid bilayer. This is illustrated in Fig. 6 which shows IH slices taken from a two-dimensional heteronuclear multiple-quantum correlation (HMQC)/NOE experiment 6° corresponding to residues that participate in the transmembrane and in-plane helices of fd coat protein. $17, a residue residing in the plane of the lipid bilayer, possesses a sharp resonance peak and has strong NOE cross-peaks to adjacent amide resonances. In contrast, A35, a residue from the transmembrane helix, possesses a broad line width and has weak or missing NOE cross-peaks to adjacent amide resonances. A highly effective way to improve the intensity of NOE cross-peaks between residues, and to reduce ~H line widths, is to label the protein uniformly with 15N and 2H56;the presence of 2H instead of ~H nuclei on side-chain and backbone carbons isolates the amide N - H hydrogens, which attenuates the interactions among hydrogens responsible for line broadening and spin diffusion. The benefits of deuteration are illustrated by comparing the spectra in Fig. 6B,C. Instrumentation

Multidimensional solution NMR experiments on peptides and proteins in micelles are feasible using commercially available spectrometers. These systems give dramatically better results with high-field (600-750 MHz) spectrometers compared to lower field (-<500 MHz) spectrometers. 60 D. Marion, P. C. Driscoll, L. E. Kay, P. T. Wingfield, A. Bax, A. M. Gronenborn, and G. M. Clore, Biochemistry 28, 6150 (1989).

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N M R STUDIES OF MEMBRANE PROTEINS

551

$17

B

I K48

C

t K48

9.0

8.0 O0m

FlG. 6. IH NMR spectra of fd coat protein in SDS micelles in solution. The slices were taken from two-dimensional IH-IH NOE/1H-ISN HMQC spectra obtained with a mixing time of 250 msec. (A) Uniformly tSN-labeled coat protein. The correlation peak for Ser-17 and its NOE cross-peaks to Ala-16 and Ala-18 are marked. (B) Uniformly ~SN-labeled coat protein. (C) Uniformly tSN- and -'H-labeled coat protein. The correlation peak for Ala-35 and its NOE cross-peaks to Gly-34 and Thr-36 are marked. The correlation peak for Lys48 also appears at this ~SNfrequency.

Dynamics It is n e c e s s a r y to describe the d y n a m i c s o f individual residues in solution N M R studies using relaxation m e a s u r e m e n t s b e c a u s e this p r o v i d e s information a b o u t the intramolecular motions and limits the magnitude o f the structure determination p r o b l e m by identifying those residues that participate in stable structures. T h e IH/~SN h e t e r o n u c l e a r N O E , along with the line widths and TI values o f both 1H and ISN r e s o n a n c e s , are effective m o n i t o r s o f b a c k b o n e d y n a m i c s . ~7,6~ ~3C relaxation p a r a m e t e r s can also be used to characterize the d y n a m i c s o f these proteins. 62'63 T h e motional properties o f the p o l y p e p t i d e described on the basis o f relaxation m e a s u r e m e n t s on m e m b r a n e proteins in micelles are c o m p l e m e n t a r y to 61 L. E. Kay, D. A. Torchia, and A. Bax, Biochemistry 28, 8972 (1989). 6., T. A. Cross and S. J. Opella, Biochem. Biophys. Res. Commun. 92, 478 (1980). 63G. D. Henry, J. H. Weiner, and B. D. Sykes, Biochemistry 25, 590 (1986).

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PROTEIN STRUCTURE

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the results from solid-state NMR experiments of proteins in bilayers, since they both identify regions of a protein participating in stable secondary structure. Comparisons between solid-state NMR and solution NMR results are particularly interesting, since it is possible to find residues that are mobile on the slow time scales of solid-state NMR powder-pattern line shapes but are immobile on the rapid time scales of solution NMR relaxation measurements. 64

Structure

As discussed in other chapters, the determination of the structures of proteins in solution by multidimensional NMR spectroscopy is straightforward when the proteins are uniformly labeled with 13C and 15N and are reasonably soluble and stable in solution. Problems arise when the proteins reorient slowly in solution, owing to their large size or aggregation. The deleterious effects of spin diffusion on homonuclear NOE measurements compound the sensitivity and resolution problems caused by the broad line widths of resonances and their limited chemical shift dispersion, especially from hydrogens bonded to a carbons. The effects of these problems are minimized by the isotopic labeling schemes and sample preparation described above. The sequential assignment of spin systems, including side-chain and a carbon proton assignments are made with a combination of experiments, especially three-dimensional total correlation spectroscopy (TOCSY)/HMQC 65 supplemented with triple-resonance 13C/15N-1H experiments 66 and selective isotopic labeling. The protein structure determination relies on the measurement of homonuclear ~H-~N NOEs in multidimensional spectra of samples uniformly labeled with 15N and 2H.3'5'56 Potentially, the secondary structure elements could be oriented relative to one another through long-range NOEs observed in micelles. In general, however, solid-state NMR experiments on selectively labeled oriented samples in bilayers are needed to find the orientations of the helices. The measurement of homo- and heteronuclear spin coupling constants in proteins provides local conformational information and an additional structural constraint, although the broad line widths of proteins in micelles limits the possibilities for making these measurements.

6~ M. J. Bogusky. G. C. Leo, and S. J. Opella, Proteins: Struct. Funct. Genet. 4, 123 (1988). 6~ G. M. Clore. A. Bax, and A. M. Gronenborn, J. Biomol. N M R 1, 13 (1991). ~'~ L. E. Kay. M. lkura. R. Tschedin, and A. Bax, J. Magn. Reson. 89, 496 (1990).

[19]

N M R STUDIES OF MEMBRANE PROTEINS

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Example of Combined Studies of Micelle and Bilayer Samples

Description of f d Coat Protein System The filamentous bacteriophage fd infects Escherichia coli and has approximately 2700 copies of a 50-residue major coat protein surrounding its D N A . 67,68 The viral coat protein is synthesized with an N-terminal signal sequence which directs it to the bacterial inner membrane 69 before being removed by cleavage with leader peptidase. When the coat protein becomes the most abundant membrane in infected cells, subunits assemble to form the outer coat of the virus particles as they are extruded through the membrane. The structure and dynamics of the membrane-bound form of the protein are being described using the approach presented in this c h a p t e r . 4'5'44'64 This protein provides an excellent system for developing methods for determining the structures of membrane proteins by NMR spectroscopy because it is a small but typical membrane protein with both amphipathic and hydrophobic helices.

Multidimensional Solution Nuclear Magnetic Resonance Experiments The determination of the secondary structure offd coat protein in SDS micelles utilizes multidimensional NMR spectroscopy. 4'5 The majority of the sequential assignments of the amide resonances were made using homonuclear JH NOE data from two- and three-dimensional NOE/HMQC experiments, as shown in Fig. 7, supplemented with information from TOCSY/HMQC experiments, and spectra of selectively 15N-labeled samples. All of the NOE data are consistent with extensive helical secondary structure in the protein; in particular, NOEs are observed between NH-NH(i, i + 1), NH-C"H(i, i + 1), NH-C~H(i, i+ l), NH-C"H(i, i + 3), and NH-C"H(i, i + 4) for many residues determined to have helical secondary structure. The IH-JH strips in Fig. 8 at tSN chemical shifts corresponding to residues S13, L14, and Q15 show the presence of NOEs between resonances from hydrogens on adjacent amide nitrogens, a carbons, and/3 carbons; these residues are part of an amphipathic helix that lies in the plane of the lipid bilayer. Residues K40, L41, and F42 also exhibit these characteristic NOEs and make up part of the hydrophobic transmembrane helix. The numerous NOEs observed for residues 13-15 67 L. Makowski, in "Biological Macromolecules and Assemblies" (A. McPherson, ed.), p. 203. Wiley, New York, 1984. 68 M. Russel, Mol. Microbiol. 5, 1607 (1982). 69 C. Chang, G. Blobel, and P. Model, Proc. Natl. Acad. Sci. U.S.A. 75, 361 (1978).

554

PROTEIN STRUCTURE

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FIG. 7. Three-dimensional (~H-~H NOE/IH-15N HMQC) solution spectrum of uniformly ~SN-labeled fd coat protein in SDS micelles in solution.

and 40-42 are between NHC"H(i, i + 3) and NHC~H(i, i + 4) sites, strongly supporting the presence of helical secondary structure for these residues. The absence of NOEs for residues 1-5 at the amino terminus of the coat protein indicates that these residues do not participate in stable secondary structure, and the strip from the NOE/HMQC experiment corresponding to G3 in Fig. 8 has no NOEs to any other residue. Residues 1-5 are highly mobile, as seen in relaxation data of the coat protein in micelle samples. 44 81.~

88.09

fi,lppm S13

94.97 1.14

90.63

fi,/ppm

91.35

91.35

93.16

Sl Ippm

[~15

O.N

0.00

0.00

7.50

~

!,,=z~

..~... r

8.115

8.95

9.04

FIG. 8. Strips taken from a three-dimensional ( H - I H NOE/IH-~SN HMQC) spectrum of uniformly 15N-labeled fd coat protein in S D S micelles in solution.

[19]

N M R STUDIES OF MEMBRANE PROTEINS

555

Solid-State Nuclear Magnetic Resonance Experiments The ~5N chemical shift powder patterns from unoriented samples of uniformly (Fig. 3A) and selectively ~SN-labeled fd coat protein in lipid bilayers (Fig. 3B) establish that the membrane-bound form of the protein has both mobile and rigid backbone sites. The amide groups of both L14 and L41 are immobile in membrane bilayers because the powder-pattern spectrum in Fig. 3B shows little evidence of motional averaging. However, the spectrum in Fig. 3A from the uniformly 15N-labeled sample has substantial isotropic intensity from the mobile N- and C-terminal residues. The 15N solid-state NMR spectra of selectively [~SN]leucine-labeled coat proteins in unoriented phospholipid bilayers and in oriented phospholipid bilayers are shown in Figs. 3B and 9B, respectively. Comparison of these spectra shows the effect of sample orientation; the two 15N resonances go from a broad, characteristically shaped powder pattern to narrow, single lines with frequencies determined by the orientation of the peptide planes with respect to the direction of the magnetic field. Comparison of the spectra in Figs. 3A and 9A also shows the effect of sample orientation, although at lower resolution because of multiple overlapping resonances in the uniformly 15N-labeled samples. However, the resonance bands occur at the positions expected for transmembrane and in-plane helices. Because the secondary structure for the two leucine residues is established as helix by the NOE data in Fig. 8, the resonance frequency

A

B

Crll z6o

6 ppm

FIG. 9. Solid-state 15N NMR spectra of fd coat protein in oriented lipid bilayer samples.5 (A) Uniformly ESN-labeledcoat protein; (B) selectively [~SN]Leu-labeledcoat protein.

556

PROTEIN STRUCTURE

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of each nitrogen site can be used to establish the orientations of the helices relative to the plane of the bilayer. The spectrum in Fig. 9B of [~SN]leucinelabeled coat protein in oriented bilayers has one line with a resonance frequency near o-iiand a second line with a resonance frequency near o- x, indicating that the hydrophobic helix spans the bilayer because the NH bond of one of the leucine residues is parallel to the direction of orientation and that the amphipathic helix is in the plane of the bilayer because the NH bond of the other leucine residue is perpendicular to the direction of orientation.

Model of Membrane-Bound Form of Coat Protein Figure 10 summarizes the results of the NMR experiments: '5 The structure and dynamics of the membrane-bound form of fd coat protein were determined by combining results from solid-state NMR experiments of the coat protein in bilayers with solution NMR results of the coat protein in micelles. The secondary structure of the protein is almost entirely helical based on the observation of NOEs among all amide resonances from adjacent residues 7-50 and the NOEs between hydrogens on nitrogen amide and a carbons three and four residues away. The orientation of the helical segments was determined by solid-state NMR experiments that showed residues in the amphipathic helix lying parallel to the plane of the bilayer and residues in the hydrophobic helix spanning the bilayer. Tyrosine-21 is located between the two helices; it is structured on short times scales but shows evidence of mobility on the slower solid-state

FIG. 10. Structural model of the membrane-bound form of fd coat protein based on NMR data. 4'5

[19]

N M R STUDIES OF MEMBRANE PROTEINS

557

NMR time scale. 5 The presence of strong N H - N H (i, i + 1) and NH-C~H (i, i + 2) NOEs and the mobility of Y21 on slow time scales suggest that it participates in a turn that connects the two helical regions. Residues 1-5 at the amino terminus of the protein are highly mobile based on a variety of relaxation data in micelle samples and motional averaging of line shapes in bilayer samples. 17'44 Residues at the carboxy teminus are also mobile in both bilayer and micelle samples~7'44; however, the observation of homonuclear NOEs indicate that helical secondary structure extends through to the C terminal on some time s c a l e s Y The features of the model of the membrane-bound form of fd coat protein are similar to those found for the membrane-bound form of Pfl coat protein, 3 in spite of the absence of sequence similarity between the proteins. The structure and dynamics of the membrane-bound forms of both fd and Pfl coat proteins were characterized by combining the results of solution and solid-state NMR spectroscopies. Both proteins consist of helical secondary structure intermixed with mobile termini and loop regions. The helical regions in fd and Pfl coat protein consist of two segments, with hydrophobic residues forming a transmembrane helix and amphipathic residues forming a helix parallel to the bilayer plane. The two proteins differ most in the extent of mobility of the segment connecting the two helices. The connecting loop residues in Pfl are highly mobile on both slow (10 .4 sec) and fast (10 -9 sec) time scales with an absence of homonuclear 1H NOE cross-peaks involving the amide resonances for these residues. In contrast, fd coat protein has homonuclear NOE crosspeaks throughout the sequence, including the residues involved in the turn connecting the two helices; there is evidence of limited motional averaging for Tyr-21.5 Prospects for Nuclear Magnetic Resonance Studies o f Membrane Proteins The overall goal of experimental NMR studies of membrane proteins is to contribute to bringing these proteins into the realm of structural biology. This involves determining the three-dimensional structures within the ordered context of membrane bilayers and integrating the structures and dynamics into models for mechanisms of action. It is hoped that the further development of experimental NMR methods will be accompanied by the identification of crystallization conditions so that NMR spectroscopy and X-ray crystallography can play complementary roles similar to those that are so successful in studies of globular proteins. The solid-state NMR methods for measuring internuclear distances that utilize magic angle sample spinning do not require oriented sam-

558

PROTEIN STRUCTURE

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pies. 46'49 This is highly advantageous because it simplifies sample prepara-

tion, and it will allow these methods to be applied in some situations where no other method can be. The selectivity and accuracy of these methods are being improved in the course of development, and they are already providing valuable structural information about membrane proteins. 22 The NMR methods that utilize uniaxially oriented samples 5°-53not only provide measurements of angles between bonds (and chemical groups) and the direction of sample orientation (and the applied magnetic field), which can be interpreted in terms of structures in much the same way that distance measurements can be, but also orientation of the structural elements relative to the plane of the bilayers. ~This is important for understanding the functions of membrane proteins, since they reside and act in the context of a membrane with an "inside" and an "outside," which is a very different situation than the more or less isotropic environment of the cell cytoplasm and periplasm occupied by globular proteins. Determination of orientations of residues and domains can only be derived from experiments on macroscopically oriented samples. The most pressing need is to develop improved methods of sample orientation for solid-state NMR spectroscopy. Although the use of a single pair of glass plates and a fiat-coil probe 38 goes a long way toward facilitating studies of proteins embedded in lipid bilayers oriented between glass plates, and there is a great deal that can be accomplished with these samples, there are limits to the extent of orientation and amount of protein that can be incorporated into the lipids. These limits can undoubtedly be reduced through additional development and refinement. However, it would be preferable to find a lipid or liquid crystalline medium that would enable the magnetic alignment of membrane proteins with their bilayer normal (and the transmembrane helices parallel) to the direction of the applied magnetic field. This direction of orientation is essential to give spectra where the spin interactions at individual sites yield single resonance lines characterized by their resonance frequencies or doublets (or triplets) characterized by the magnitudes (and asymmetry) of their splittings. 53Unfortunately, most of the magnetically orientable media orient perpendicular to the field, 25'26 which causes many complications in the spectra, including almost complete loss of resolution if powder patterns are present, and their interpretation. Samples of membrane proteins in magnetically orientable liquid crystals would allow the use of solenoidal coils and larger samples, improving essentially all aspects of the spectroscopic experiments, and some progress has been reported. 32-33 Many of the initial structural studies of peptides and proteins in oriented samples used specifically L55 or selectively 3-5'5° ~SN-labeled samples

[19]

N M R STUDIES OF MEMBRANE PROTEINS

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prepared by automated solid-phase peptide synthesis or expression in bacteria. However, it would be desirable to reduce or eliminate the use of specifically or selectively labeled samples. Unlabeled or uniformly labeled samples would be preferable. The obstacles to their use is the availability of nuclear spin interactions for analysis and the resolution among overlapping resonances. Uniformly ~SN-labeled proteins are suitable for both solidstate N M R 7° and multidimensional solution N M R 71 experiments, and the resolution can be improved by going to three- or higher dimensional experiments. Three-dimensional solid-state NMR experiments rely on the various 1H and 15N chemical shift and dipolar interactions present in the amide backbone sites; depending on the choice of experiment, structural information and resonance assignments can be obtained. 72,73At high resonance frequencies the ~H amide chemical shift interaction becomes a useful parameter present in natural abundance capable of providing both resolution and orientational information, since the tensor for the amide site has been determined. TM Also present in natural abundance is the 14N quadrupole interaction, which is accessible with ~4N overtone spectroscopy. 75'76 By combining high-dimensional experiments with the spin interactions available in JH and 14N sites, it is possible to study proteins by solid-state NMR spectroscopy without labeling. The development of multidimensional solution NMR spectroscopy of proteins in micelles will be very similar to that for larger globular proteins, since the limiting factor, rotational correlation time, is the same in both cases. The use of multiply labeled protein samples and carefully prepared micelles is essential. These samples are acutely sensitive to field strength, and as high-field spectrometers become available, these experiments will become more widely applicable. In summary, NMR spectroscopy has been successfully applied to the study of selected membrane peptides and proteins. Therefore, there is good reason to be optimistic that as the samples and spectroscopic methods are improved, NMR spectroscopy will become generally applicable to membrane proteins. It is also worth keeping in mind the current status of structural studies of membrane proteins. Because there is such a lack 70 T. A. Cross, J. A. DiVerdi, and S. J. Opella, J. Am. Chem. Soc. 104, 1759 (1982). 71 M. J. Bogusky, P. Tsang, and S. J. Opella, Biochem. Biophys. Res. Commun. 127, 54O (1985). 7_, B. S. Arun Kumar and S. J. Opella, J. Magn. Reson. 95, 417 (1991). 7~ B. S. Arun Kumar and S. J. Opella, J. Magn. Reson. Ser. A 101, 333 (1993). 74 R. Gerald, T. Bernhard, U. Haeberlen, J. Rendell, and S. J. Opella, J. Am. Chem. Soc. 115, 777 (1993). 7s R. Tycko and S. J. Opella, J. Chem. Phys. 86, 1761 (1987). 76 p. L. Stewart, R. Tycko, and S. J. Opella, J. Chem. Soc., Faraday Trans. 1 84, 3803 (1988).

560

PROTEIN STRUCTURE

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of information about the structures of these proteins, knowledge of the basic organization of the secondary structure and its location relative to the membrane is, by itself, valuable. And this information can be gained from studies of uniformly 15N-labeled samples using currently available methods and instruments, since it is so easy to differentiate transmembrane from in-plane helices qualitatively. It is then a matter of spectroscopic refinement to take these initial results to three-dimensional structures. NMR spectroscopy has the potential to open an entire area of structural biology with thorough studies of membrane proteins. Acknowledgments We thank B. Bechinger, J. Gesell, K. G. Valentine, and T. Le for participation in the construction of instruments and the experimental studies and L. Gierasch, M. Montal, J. Tomich, and M. Zasloff for samples and discussions which contributed to the development and application of the NMR methods for studying membrane proteins described in this chapter. This research has been supported by Grants A120770, GM29754, GM24266, and RR05976 from the National Institutes of Health.