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P1: PSA/spd P2: ARS/ARY QC: PSA/anil T1: PSA

April 14, 1999 19:44 Annual Reviews AR083-10

Annu. Rev. Biophys. Biomol. Struct. 1999. 28:235–68Copyright c© 1999 by Annual Reviews. All rights reserved

SOLID-STATE NUCLEARMAGNETIC RESONANCEINVESTIGATION OF PROTEINAND POLYPEPTIDE STRUCTURE

Riqiang Fu1 and Timothy A. Cross1,2

1Center for Interdisciplinary Magnetic Resonance at the National High MagneticField Laboratory and2Department of Chemistry, Institute of Molecular Biophysics,Florida State University, Tallahassee, Florida 32310;e-mail: [email protected], [email protected]

KEY WORDS: orientational constraints, oriented samples, distance constraints, magic anglespinning, torsional constraints

ABSTRACT

Solid-state nuclear magnetic resonance (NMR) is rapidly emerging as a suc-cessful and important technique for protein and peptide structural elucidationfrom samples in anisotropic environments. Because of the diversity of nucleiand nuclear spin interactions that can be observed, and because of the broadrange of sample conditions that can be studied by solid-state NMR, the poten-tial for gaining structural constraints is great. Structural constraints in the formof orientational, distance, and torsional constraints can be obtained on proteinsin crystalline, liquid-crystalline, or amorphous preparations. Great progress inthe past few years has been made in developing techniques for obtaining theseconstraints, and now it has also been clearly demonstrated that these constraintscan be assembled into uniquely defined three-dimensional structures at high res-olution. Although much progress toward the development of solid-state NMR asa routine structural tool has been documented, the future is even brighter withthe continued development of the experiments, of NMR hardware, and of themolecular biological methods for the preparation of labeled samples.

2351056-8700/99/0610-0235$08.00

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236 FU & CROSS

CONTENTS

PERSPECTIVES AND OVERVIEW. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236

ORIENTATIONAL CONSTRAINTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239Specific Site Isotopic Labeling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241Amino Acid Specific Labeling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243Uniform Labeling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245

DISTANCE CONSTRAINTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246Homonuclear Distance Measurements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249Heteronuclear Distance Measurements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252

TORSIONAL CONSTRAINTS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256

FUTURE DIRECTIONS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259

PERSPECTIVES AND OVERVIEW

Fifteen years ago, a paper was published that demonstrated that high-resolutionstructural constraints could be obtained by solid-state nuclear magnetic reso-nance (NMR) of proteins in hydrated but anisotropic environments (15). Thisreview documents the progress made since that early paper, with a focus onthe very recent accomplishments in this field. The potential of this approachhas been rapidly evolving through the development of new types of constraints,the collection of many constraints simultaneously, and the determination ofthe first three-dimensional structure for the Protein Data Bank (accession code#1mag).

Knowledge of high-resolution protein structure is severely biased towardwater-soluble proteins. Membrane and structural proteins, as well as proteinsthat exist in very-high-molecular-weight complexes, are not well represented inthe data banks. Structural proteins are often long filaments that aggregate, mak-ing crystallization nearly impossible for X-ray crystallography. Similarly, suchcharacteristics make solution NMR methods nonviable. Although the struc-ture ofBombyx morisilk fibroin was one of the first protein structures solved(72), the fiber diffraction characterization represented a low-resolution struc-ture that has not been substantially improved until recently (18). Work withβ-amyloid peptides that form complex aggregates represent another examplewhere the standard protein structural methods are not successful because ofan inability to crystallize and/or solubilize the protein in a structurally relevantenvironment. Despite some recent spectacular successes by diffraction (19, 85),membrane proteins pose similar problems for both solution NMR and X-raycrystallography. Membrane proteins represent as much as one third of theMy-coplasma genitaliumproteome (25). With so many critically important proteinsthat are exceptionally difficult to crystallize or solubilize into isotropic solution,a new structural approach has been needed (12, 17, 83).

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SOLID-STATE NMR OF PROTEINS 237

Solid-state NMR has the inherent ability to detect single atomic sites and,hence, the potential to yield high-resolution data. At high field, sensitivity isvastly improved, to the extent that reasonable amounts of isotopically labeledprotein are required for obtaining high-quality spectra. The restriction for solu-tion NMR, that samples must be isotropically tumbling, does not apply to solid-state NMR: for solid-state NMR, the more rigid the samples the better. Indeed,this reflects the distinction between solid-state NMR (observation of anisotropicsystems) and solution NMR (observation of isotropic systems). Consequently,the “correlation time problem” so frequently discussed as a molecular weightlimit for solution NMR does not apply to solid-state NMR (83). The molecularweight range that can be studied by NMR is nearly without bounds, hence stud-ies of silk fibroin (18), of the 16-MDa filamentous virus fd (94), and of colicinE1 P190 in planar lipid bilayers (58).

There are many approaches for gaining structural insights into proteins fromNMR. Long before the NOESY experiment was developed for solution NMR,structural information was being sought from spectra of protein solutions. Sim-ilarly, in solid-state NMR there are many approaches for gaining structuralinsight. Characterizations of pKas, hydrogen-deuterium exchange, and inter-pretation of isotropic chemical shifts are a few such approaches. This review,however, focuses on three types of structural constraints that have the greatestdemonstrated capacity for playing a major role in the elucidation of completethree-dimensional protein structures: orientational, distance, and torsional con-straints. As the interpretation of isotropic chemical shifts and the inherent chem-ical shift anisotropy becomes a more precise science, it is entirely possible thatconstraints from these observations will become much more important. Indeed,very significant progress in this arena has been made recently (40, 41, 93, 111).Hydrogen-deuterium exchange has also developed into an important tool, fromthe work of Harbison et al (39) to the recent folding studies of a membranebound polypeptide (9).

However, currently the primary structural tools for solid-state NMR spec-troscopists are the measurement of specific site orientations with respect to thelaboratory frame from aligned samples and, using unoriented samples, the mea-surement of intramolecular distances and the measurement of specific torsionangles. Orientational constraints are observed in samples that have a uniqueorientation with respect to the magnetic field axis of the NMR spectrome-ter. The data from an orientation-dependent nuclear spin interaction constrainsthe orientation of a specific molecular site with respect to the axis of align-ment. By obtaining numerous constraints, all with respect to the same axis,three-dimensional structures can be achieved. Distance constraints that definethe relative separation of two atoms can be obtained through observation of

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238 FU & CROSS

residual dipolar interactions. Both homonuclear and heteronuclear interactionscan be observed with considerable precision. As is routine in solution NMR,three-dimensional structures can be solved by distance constraints. Torsionalconstraints are defined through the observation of the relative orientation ofspin interaction tensors in adjacent sites within the protein. Such constraintslead directly to the definition of the structural torsion angles, the ultimate goalfor defining three-dimensional structure.

The assembly of three-dimensional structures is similar to solution NMRwhere distances and torsional constraints are involved; however, orientationalconstraints are unique in this regard. These latter constraints are absolute and in-dependent, unlike the torsional and distance constraints, which are relative anddependent constraints. The independent nature of the orientational constraintsmeans that the errors associated with individual constraints do not sum whenmultiple constraints are used for defining protein structure. Moreover, becausethe constraints are very precise, the search of conformational space consistentwith the constraints is challenging because of the high-penalty barriers betweenpenalty function minima (57). Although each of these types of constraints hasadvantages, in combination there is great synergy. Orientational and torsionalconstraints can determine the relative orientation of molecular groups, and fororientational constraints this applies even to groups vastly separated in the pro-tein, but neither constraint is appropriate for precisely defining the separation ofthese groups, a task directly solved by distance constraints. Conversely, evenprecise distance measurements may leave the relative orientation of groupspoorly defined. To date, few examples exist in the literature where combinationsof structural constraint types have been used in solid-state NMR (6, 9, 30). Un-til the spectroscopy for each of these constraint types becomes more routine,such papers will be rare. Moreover, the number of constraints typically de-veloped by solid-state NMR for any one structure has been relatively small;however, the precision of the constraints is much higher than in solution stateNMR. Consequently, even with relatively few constraints, high-resolution struc-ture is achievable.

It is important to recognize that for characterizing protein conformation, thedetermination of the torsion angles within the protein is adequate for the struc-tural solution. Even for high-resolution crystal structures the elucidation of bondangles and bond lengths is rarely obtained from the crystal data. Consequently,the structural problem from this solid-state NMR view does not involve theindependent characterization of the relative position of all atoms. Rather, it isthe characterization of torsion angles that connect such relatively rigid struc-tural elements as an indole group, a methylene, or a peptide plane. Even in thislatter example, the plane could be considered as two halves separated by theω

torsion angle.

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ORIENTATIONAL CONSTRAINTS

Orientational constraints are derived from observations of a wide range ofanisotropic nuclear spin interactions, such as chemical shifts, homo- and hetero-nuclear dipolar interactions, and quadrupolar interactions. The quality of theconstraints is dependent on many parameters: how well the orientation of thespin interaction tensors are known with respect to the molecular frame, howwell the samples are aligned, how well the dynamics are characterized for thesite, relaxation rates of the observed nuclei, and the specific orientation ofthe site. These parameters can be characterized independently, and many ofthem have only a modest impact on the accuracy of constraint interpretation.Consequently, the precision and accuracy of these constraints can be very high.

Some interaction tensor orientations, such as the dipolar interactions betweencovalently attached nuclei, are accurately known whereas a deuteron bound toa nitrogen atom or many chemical shift tensors are less well known. Amide15Nchemical shift tensors have been extensively studied and although the tensorelement magnitudes vary by 10–20%, the tensor orientation with respect to themolecular frame is invariant except for glycine. The glycine tensor orientationdiffers by 4–6◦ compared with the other amino acids (70, 80). Tensor elementmagnitudes can be characterized through observation of the unoriented spec-trum of a single-site–labeled peptide. It is important to realize that differentcrystal forms of the same peptide can yield substantially different tensor el-ement magnitudes (44). Consequently, it is best to characterize tensors notfrom model compounds, but from the compound of interest in the environmentof interest. Tensor orientations have been determined using single crystals ofmodel compounds, as well as through a characterization of the relative orien-tation of a known tensor (e.g. dipolar interaction) to a less-well-defined tensor(e.g. chemical shift). This can also be done for peptides in the environmentof interest (100). As more tensors are characterized and more orientationalconstraints interpreted, the variability in the tensors will be realized and theneed for numerous tensor characterizations reduced.

Much effort has gone into the improvement of sample alignment. A dis-persion of molecular orientations with respect to the magnetic field axis willbroaden the observed resonances because the resonance frequencies reflect thisorientation with respect to the axis. Consequently, a broad resonance will de-crease the accuracy and precision with which the molecular orientation canbe determined. However, these NMR studies have a tremendous advantage inthat they are performed in a high magnetic field, where the anisotropy of thediamagnetic or paramagnetic (for some samples) susceptibility can aid samplealignment. This magnetic influence was responsible for the first protein orien-tational constraints observed through aligned microcrystals of myoglobin (82)

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240 FU & CROSS

and liquid crystalline filamentous viral solutions (15). Even in bilayer prepara-tions where mosaic spreads as small as±0.3◦ have been observed, it is clear thatshear alignment between glass plates is not solely responsible for the excellentalignment (10). Prosser et al (87) took advantage of paramagnetic susceptibilityto reorient lipid disc-like aggregates known as bicelles (90) so that the vector,perpendicular to the bilayer surface, is parallel to the magnetic field axis. Thediamagnetic susceptibility of the lipids alone is such that the normal aligns at90◦ with respect to the field, but with the addition of paramagnetic ions, theweak diamagnetic term is dominated by the paramagnetic susceptibility. Suchpreparations are actively being pursued as orienting matrices for membraneproteins.

Characterization of molecular dynamics is one of the major strengths of solid-state NMR. When samples have significant dynamic flexibility, it is essentialthat the influence of structure on the observed spectra be isolated from the in-fluence of dynamics. Wide-line spectroscopy of unoriented samples can lead todetailed characterization of the molecular motion, especially when combinedwith temperature dependence. Below 200◦K, almost all motions around thetorsion angles cease. Raising the temperature above 200◦K permits the assess-ment of motional averaging of the anisotropic spin interactions by such mo-tions. These characterizations differ from relaxation studies because all motionsfaster than the interaction magnitude (e.g. 104 Hz for some chemical shift inter-actions) influence the spectra independent of motional frequency (48), whereasthe influence on relaxation properties by molecular motion has a severely non-linear frequency dependence. Consequently, it is possible to define the axis ofmolecular motion, and its amplitude with wide-line NMR and with relaxationrates define the frequency of the molecular motion without assuming a motionalmodel (78).

If the resonances being observed as a structural constraint suffer from efficientT2 relaxation, the line will be substantially broadened, reducing the precisionwith which the orientation of the site can be assessed. Fortunately, there arenumerous spin interactions to choose from and severe problems can be avoided.Finally, the orientation of the tensor with respect to Bo influences the accuracywith which the orientation of the site can be determined. For instance, thedipolar interactions have an orientational dependence such that the observeddipolar splitting,1νobs, equals the interaction magnitude,ν‖, multiplied by theorientation dependence:

1νobs= ν‖(3 cos2 θ − 1).

θ is the angle of the unique tensor axis with respect to Bo. For an N-H bond,the unique axis is aligned parallel to the covalent bond. For this interaction,the difference in1νobs for θ = 0◦ and 5◦ is 240 Hz, whereas the difference for

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θ = 40◦ and 45◦ is 2790 Hz. Although the former difference is on the border ofdetectability, the angles determined in the vicinity of 40◦ have a much smallererror bar.

Specific Site Isotopic LabelingThe advent of peptide synthesis has had a profound influence on the devel-opment of orientational constraints, because in this way the above parameterscan be extensively characterized (13). Utilizing this technology and the morethan 120 orientational constraints observed from15N and13C chemical shifts,from 15N-1H, 15N-2H, and15N-13C dipolar interactions, and from2H quadrupo-lar interactions, it has been possible to solve the structure of the polypeptidegramicidin A (gA) (55, 57). This peptide has an alternating sequence of 15amino acid residues withD andL stereochemistry. The structure is aβ-strandwith all side chains on one side, forcing the strand into a helix. A mono-valent cation-selective channel is formed by a dimer of this structure acrosslipid bilayers. Many laboratories have contributed to the characterization of thetensors, development of the experimental constraints, and characterization ofthe dynamics (e.g. 48, 59, 86, 97). The interpretation of the orientational con-straints is complicated because of the cos2 θ dependence leading to multipleorientational solutions for each observable. Consequently, despite the precision,even accuracy of the constraints, there are ambiguities that must be resolved.Multiple constraints often eliminate some of these ambiguities, as does the re-stricted covalent geometry of the structural elements. With 120 constraints for37 structural elements in this 15–amino acid polypeptide, it has been possibleto uniquely define the orientation of each structural element with respect to Boand the channel axis of this peptide. The assembly of these orientations into astructure shows both the weaknesses and the strengths of this solid-state NMRapproach. Gramicidin has 6.5 residues per turn, and if the errors of each peptideplane orientation accumulated in assembling the structure, there would be con-siderable ambiguity as to the hydrogen bond pattern, e.g. between residue 1 and6, 7, or 8. Instead, the hydrogen bond pairs are all uniquely defined within 1Aroot mean square deviation (rmsd) of ideal hydrogen bond geometry, illustratingthe advantage of using absolute structural constraints. However, in assemblingthe individual side chains, some van der Waals overlap is generated betweenside chains, similar to the 1-A error in hydrogen bond distances described above.Consequently, the initial structure is not perfect. A refinement against all theexperimental constraints, hydrogen bond distances, and CHARMm (4a) energyresults in a unique structure, with each torsion angle defined to within a fewdegrees and virtually all the constraint ambiguities solved. An example of thehigh-precision orientational constraints is shown in Figure 1 for a 5-fluoro-d4-Trp13–labeled gA preparation in dimyristoylphosphatidylcholine bilayers. The

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242 FU & CROSS

Figure 1 (Left) High-resolutional orientational constraints derived from2H nuclear magnetic res-onance (NMR) spectra of 6F-d4-Trp13–labeled gA in uniformly aligned lipid bilayers of dimyris-toylphosphatidylcholine (1:8 molar ratio, 40◦C). The19F-2H dipolar coupling is observed on theinnermost quadrupole splitting. Fluorine has been incorporated to assess the influence of altereddipole moments on the channel conductance. (Right) High-resolution structure of the gA channelshowing a single file column of water molecules supported by the narrow pore spanning the lipidbilayer. The structure has been characterized solely based on solid-state NMR-derived orientationalconstraints. The structure is aβ strand of amino acids with alternatingD andL stereochemistrythat places all side chains on one side of a strand, hence forming a helix with all side chains facingthe lipid environment.

fluorinated indole rings are being studied to further characterize the influenceof the indole dipoles on the potential energy barrier for cation transport at thecenter of the bilayer (5, 47). The refined structure of the gramicidin channel isalso shown in Figure 1.

Single-site labels have also been used to study the 26–amino acid peptidemelittin (96) and the 25–amino acid transmembrane peptide from M2 pro-tein (60). Melittin is a bee venom toxin that forms a voltage-dependent anion-selective channel in lipid bilayers. The peptide is known to be predominantlyα-helical. The 10 backbone carbonyl13C labels incorporated individually intomelittin were used to show that the peptide adopts a helical conformation and atransbilayer orientation in membranes. Although crystal studies have suggestedthat the peptide has two helical segments with an angle of 120◦ between them

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(102), there is little evidence of a different orientation for the two helical seg-ments from the orientational constraints. Therefore, the solid-state NMR dataare more consistent with the solution NMR results in micelles, where the angleis closer to 160◦. M2 protein from influenza A virus is a proton channel in theviral capsid that plays a key role in infection. Although the protein has only 97amino acid residues, the channel structure is formed by a tetramer of the pro-tein. The nine15N chemical shifts observed for the single M2 transmembranepeptide (M2-TMP), which is known to beα-helical, define the helical orienta-tion with respect to the bilayer normal as 33◦, assuming an ideal helix. Moreover,it is shown that the known tetramer is symmetric or pseudosymmetric, becausemultiple resonances for individual labels are not observed. If it is also assumedthat the hydrophilic residues are on the interior of the four-helix bundle, thenthe bundle must be left-handed.

In a related approach, Ulrich et al have specific-site–labeled retinal boundto bacteriorhodopsin (107) to determine details of the bound ligand conforma-tion. It was further shown that a change in retinal orientation occurs betweenthe initial and M states of the complex (106). Similarly, 11-cis retinal bound torhodopsin has been characterized (34). These studies illustrate the ability ofsolid-state NMR to characterize the bound conformation of ligands to mem-brane receptors.

Amino Acid Specific LabelingMany of the initial protein structural studies by solid-state NMR were conductedby uniformly labeling an amino acid type through bacterial cultures. Manyconstraints obtained for the 50–amino acid major coat protein of the filamentousvirus, fd, were shown to be consistent with theα-helical “shingling” of the virion(16, 94).

Recent studies by Demura et al (18) onBombyx morisilk fibroin haveled to a significant refinement of the original fiber diffraction of the protein(72). This protein is dominated by the repeating sequence of Gly-Ala-Gly-Ala-Gly-Ser. Through labeling with15N and13C1,

15N-1H and15N-13C dipolarinteractions, as well as chemical shifts, have been observed from oriented sam-ples. Furthermore, because the silk fibroin does not undergo axial reorientation,the orientation dependence of the sample can be recorded for the asymmetricchemical shift tensors, providing a unique solution for the orientation of thesetensors with respect to the fiber axis (Figure 2). With these constraints, specificbond orientations were determined for the Ala and Gly residues. By combiningthese solid-state NMR results with the most reliable constraints from X-ray fiberdiffraction, the orientation of the major axis of the unit cell relative to the fiberaxis, the backbone torsion angles were precisely determined. As structural bi-ology focuses on challenging molecular structures, this illustrates an important

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244 FU & CROSS

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paradigm: The ability to use multiple types of constraints from diverse tech-niques will be important for solving structures of the highest complexity and atthe highest possible resolution.

Simmons et al (95) have labeled spider dragline silk with d3 Ala. Excellentorientation was achieved and the results were interpreted in terms of highlyoriented and weakly oriented domains, consistent with two crystalline domainsembedded in a glycine-rich amorphous domain.

Uniform LabelingTo make this structural approach practical for membrane proteins, it is necessaryto obtain more than a few constraints at a time, hence uniform15N and13C label-ing. With this comes the challenge of resolving the numerous resonances andassigning them. The development of the PISEMA (polarization inversion withspin exchange at the magic angle) experiment greatly improves the resolutionof static solid-state chemical shift–dipolar correlation spectra (110). Marassiet al recorded PISEMA spectra of uniformly15N-labeled fd coat protein (50amino acids) (71) and of the colicin E1 P190 protein (190 amino acid residues)(58). A majority of the resonances are resolved in these spectra (the colicin E1P190 spectrum is shown in Figure 3), but an additional spectral dimension hasalso been demonstrated, thereby gaining even more resolution (88). This use ofthe1H chemical shift dimension has been illustrated using the fd coat protein(83). These15N spectra even without assignments provide useful informationfor membrane-bound proteins. Resonances with near maximal N-H dipolar cou-pling and maximal chemical shift suggest anα-helix aligned near parallel to thebilayer normal. Resonances with half-maximal N-H dipolar coupling and mini-mal chemical shifts suggestα-helices parallel to the bilayer surface. Clearly, theassignment of these resonances is an essential next step. Homonuclear spin dif-fusion is a likely choice that has been demonstrated on model peptides (14, 89).Figure 4 shows15N homonuclear spin diffusion between selected sites in gram-icidin oriented in lamellar-phase lipid bilayers (64). Although experimentaldevelopment is still required, both spectral resolution and homonuclear spindiffusion appropriate for resonance assignments have been demonstrated.

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 2 Solid-state13C nuclear magnetic resonance spectra of an oriented block of [13C1]Glysilk fibroin fibers (Bombyx mori) observed as a function ofβL, the angle between the fiber axisand the magnetic field direction. The best-fit theoretical line shapes were obtained with the Eulerangles (α = 90 ± 5◦, β = 22 ± 5◦) that relate the fiber axis to the chemical shift tensor. Sucha unique definition of the asymmetric tensor orientations are not possible with just theβL = 0◦orientation. The well-characterized Euler angles are achieved despite a considerable mosaic spreadof 11◦ in fiber axis orientation. (TMS) Tetramethylsilane [Reprinted with permission from Demuraet al (18).]

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246 FU & CROSS

Figure 3 A two-dimensional PISEMA spectrum of uniformly15N-labeled colicin E1 P190in oriented and fully hydrated lipid bilayers of dimyristoylphosphatidylcholine and dimyris-toylphophatidylglycerol. The sample contained just 6 mg of protein and was observed at 28◦Cwith a 15N frequency of 70.97 MHz. A dipolar slice at 242 ppm is shown (left), and the spectralregion for alpha helices aligned parallel and perpendicular to the bilayer surface are indicated (top).[Reprinted with permission from Kim et al (58).]

DISTANCE CONSTRAINTS

The magnitude of nuclear dipole-dipole (DD) interactions between two spins isproportional to their gyromagnetic ratios and the orientation of their internuclearvector with respect to the applied magnetic field and is inversely proportionalto the cube of their through-space distance. Direct measurements of the dipo-lar couplings thus lead to distance constraints between nuclear spins, provid-ing one of the most universal approaches to accurately determining molecular

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structures. In contrast to solution state NMR, where the DD interactions areusually averaged by fast molecular motions so that they can only be detectedthrough cross relaxation or nuclear Overhauser effects (75), solid-state NMRexperiments enable the direct measurement of dipolar couplings and, thus, ofdistance constraints between spin pairs. In unoriented powder samples, dipolarinteractions lead to typical powder patterns, which allow determination of in-ternuclear distances in a straightforward manner. Unfortunately, the existenceof other interactions, such as the chemical shift anisotropy (CSA) and DD in-teractions with abundant nuclei such as1H and14N, tends to obscure the moreinformative homo- and heteronuclear dipolar interactions between dilute spins.

A powerful technique to yield high-resolution chemical shift isotropic NMRspectra from unoriented samples is magic angle spinning (MAS), in which thesample is spun around the axis tilted atθm = cos−1(1/

√3) from the applied ex-

ternal magnetic field. MAS averages the broadening from both the CSA as wellas weak dipolar couplings between dilute spins while strong DD interactionswith abundant nuclei are removed by irradiation at the resonance frequency ofthe abundant nuclei, providing solution-like high-resolution solid-state NMRspectra of dilute spins. High-resolution solid-state MAS NMR spectroscopyhas become an important tool for structural studies of a variety of biopolymers,including polypeptides and proteins.

A significant feature of solid-state MAS NMR is that although the anisotropicinteractions containing rich structural information (for example, CSA representsthe distribution of electrons around nuclei, and dipolar interactions are related tointernuclear distances) are suppressed by the mechanical spinning, they can beselectively recovered by spectroscopic manipulations. Extensive efforts havebeen focused on how to interfere with the effect of sample spinning on theseinteractions while utilizing the advantage of high-resolution spectra, therebyallowing for the precise and accurate measurement of individual interactions ofinterest. The weak DD couplings between dilute spins are of particular inter-est, because the dilute spins such as carbon, nitrogen, and oxygen are essentialelements for constructing the backbone of polypeptides and proteins, funda-mental to structural determination of these biopolymers. Many experimentaltechniques have been developed to prevent these weak dipolar couplings frombeing averaged out by MAS, so that precise measurements of long-range inter-nuclear distances can be obtained, providing a way to determine the secondarystructure of polypeptides and proteins.

Generally, with caution taken, the precision and accuracy of distance con-straints can be very high. Measurement of distance constraints by solid-stateNMR spectroscopy usually requires labeling with rare spin isotopes such as15N and13C at specific sites of interest in order to arrange for DD interactionsto be present and observable, thus providing the desired distance constraints

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248 FU & CROSS

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between these specific sites. However, the isotopic labels also increase theprobability of intermolecular dipolar couplings with neighboring molecules(74), particularly when long-range distances are expected or peptides aggre-gate (6, 62, 76, 98). Dilution of the isotope labels is thus required in order toattenuate the intermolecular effect so as to obtain accurate distances (6). Signif-icantly, the dilution effect allows for a calibration of these intermolecular influ-ences, thereby providing a way to access the packing arrangement. Moleculardynamics is another factor that could seriously deteriorate the accuracy of dis-tance constraints when such motions partially average the dipolar coupling. Forinstance, the measured distance between13C and15N in alanine by NMR islonger than that obtained by X-ray, owing to the fast motion in the amino group(37). Molecular vibrations also affect the accuracy of distance measurements(50). Lowering sample temperature is an effective way to minimize the effectof molecular motion, although it may induce conformational rearrangements,as in the case of enkephalins (54). On the other hand, the motionally reduceddipolar coupling may also be used (a) to characterize the orientation of the inter-nuclear vector with respect to the motional axis if the magnitude of the dipolarcoupling can be determined independently (42), or (b) to determine the dipolarcoupling if the orientation of the internuclear vector with respect to the motionalaxis can be obtained in separate experiments (9).

Distance measurements by solid-state NMR have been increasingly used tocharacterize three-dimensional structures. Depending on the source of dis-tance information, experimental techniques for distance measurements can bedivided into two general categories: homonuclear and heteronuclear dipolarrecouplings.

Homonuclear Distance MeasurementsRotational resonance (R2) spectroscopy is an important method for measur-ing homonuclear distances between two isolated spins, such as13C, that havedifferent isotropic chemical shifts. R2 is a resonance phenomenon achievedwhen the time dependence induced by sample spinning interacts with internalinteractions such as chemical shifts and DD interactions. When the chemicalshift difference between two spins matches a multiple of the sample spinningspeed—i.e.1ωiso = nωr, where1ωiso is the chemical shift difference,ωr is

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 4 Two-dimensional15N-15N spin diffusion experiment for three15N-labeled sites in thegramicidin A channel structure uniformly aligned with respect to the magnetic field axis: Gly2,Val8, and Trp9 (64). Although Gly2 is removed significantly in terms of the primary sequence,this helical structure has 6.5 residues per turn, and hence Gly2 is close to both Val8 and Trp9. Thecross-polarization pulse sequence involves mixing of the1H-coupled15N magnetization on the Zaxis for 5 s, approximately equal to the15N T1 relaxation time.

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250 FU & CROSS

the spinning speed, andn is an integer—this rotational resonance significantlyenhances the dipolar exchange rate between the two spins, which is attenuatedby MAS. The enhanced dipolar exchange rate leads to spectral line broadeningand splitting in one-dimensional spectra (79) if the homonuclear coupling isrelatively strong, such as that between two directly bonded nuclei, contain-ing structural information about the internuclear distance (66, 108). When thehomonuclear dipolar coupling is weak, it is necessary to generate a longitudinalpolarization difference between the two spins by selectively inverting one ofthe two homonuclear resonance lines in order to monitor their subsequent lon-gitudinal magnetization exchange between the two spins as a function of time.The trajectory of the population difference may then be simulated to yield anaccurate internuclear distance up to 6A for 13C-13C pairs. Depending on theinteger numbern, the trajectory is also related to other parameters, such as thechemical shift tensors of the two spins, relative orientations of chemical shiftand dipolar tensors, and the zero-quantum relaxation time (T2

ZQ), which can beindependently measured or estimated. R2 experiments have been widely usedto measure the internuclear distances in protein structures.

Griffin and coworkers (6, 62, 98) have put great effort into characterizing thecomplete structure of theβ-amyloid peptide fibril (β32-42) by applying the R2

technique to13C-13C pairs systematically labeled in the peptide chain. Thispeptide is derived from theβ-amyloid protein that is the primary constituentof the amyloid plaques, associated with Alzheimer’s disease (AD). Based onthe distance constraints from intramolecular13C-13C pairs in combination, withknowledge of the13C chemical shift frequencies and individual absorptionfrequencies (amide I absorption band of 1660–1650 cm−1) by isotope-editedinfrared spectroscopy, an initial structural analysis for theβ34-42 amyloid wasobtained. It was also shown that the intermolecular couplings severely affectedthe observed R2 exchange curves, as demonstrated in Figure 5a. Three typicalselectively labeled13C pairs in the peptide chain are indicated byI, II , andIII forintramolecular distances. Their R2 exchange curves with 100% labeled peptideand 1:5 isotope diluted peptide are also shown to illustrate the intermoleculareffects. No effect of isotope dilution is observed for the R2 exchange curve(Figure 5aI ). On the other hand, the R2 exchange curve (Figure 5aIII ) resultssolely from the intermolecular distance, because the R2 exchange for the 1:5isotope dilution is entirely abolished. Both intra- and intermolecular distancesare responsible for the R2 exchange curve (Figure 5aII ) in the 100% labeledsample. The results are consistent with an antiparallelβ-sheet structure, asshown (Figure 5b). A set of constraints from the distance measurements elim-inates many of the structures from a library generated by molecular dynamicssimulated annealing calculations with energy minimization. The center regionof the peptide is still less clear. The earlier distance measurements between the

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Figure 5 (a) Three typical magnetization exchange results obtained from R2 experiments forβ-amyloid peptide fibril (β32-42) showing the effect of isotope dilution. (I, II , III ) Correspondto labels onright. (Solid circles) The results for the 100% doubly labeled samples; (open circles)results for the 1:5 diluted samples. (b) The isotope-labeled pairs indicatedI, II , and III for theintramolecular distances andI

∗(not shown),II

∗, andIII

∗for intermolecular distances. [Reprinted

with permission from Lansbury et al (62).]

α-carbon of Gly37 and carbonyl carbon of Gly38, both of which are located inthe center region, were not able to provide a clear distinction between thecisortransconfiguration for the Gly37-Gly38 amide bond (62, 98). It is believed thatthe labeled sample was not dilute enough to attenuate the intermolecular effect.Recent spin-echo experiments andn = 2 R2 measurements clearly suggestthat the Gly37-Gly38 peptide bond is in thetransconfiguration, rather than thepreviously reportedcisbond (6).

Although R2 experiments have been extremely successful, the R2 condition isnarrow and tends to be impractical to fulfill in some cases, particularly where thechemical shift difference is small, requiring slow spinning speeds and yieldinglow-sensitivity spectra with many spinning sidebands. Many improved tech-niques have been developed. Terao and coworkers (99, 101) proposed an R2TR(rotational resonance in a tilted rotating frame) technique that extends the R2

from the rotating frame to the double tilted rotating frame by off-resonance irra-diation. The rotational resonance condition in the tilted rotating frame becomesthe difference, or the sum of amplitudes of two effective fields, rather than thechemical shift difference, as in conventional R2, is equal tonωr. The former canbe used for scaling down the large chemical shift difference and the latter forenlarging the small chemical shift difference. It is successfully demonstrated

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252 FU & CROSS

that the resonance condition can be switched with complete control by the pulsesequence, allowing different spectroscopic manipulations within the sequence(52). Rotational resonance tickling (R2T) (7) by ramping the radio frequency(RF) field through the R2 condition greatly reduces the dependence of spin dy-namics on zero-quantum parameters. Moreover, broadband homonuclear dipo-lar recoupling techniques are designed for recoupling dipolar couplings of spinsacross a broad range of chemical shifts and have been accomplished by severalgroups. An attractive advantage of these broadband recoupling techniques is toallow correlation spectra in solid-state systems with uniformly labeled isotopicspins (e.g.13C), akin to NOESY and TOCSY spectra in solution NMR, to be ob-tained, providing connectivity of the spin networks (1, 2, 20, 81). However, fewstructures of uniformly labeled samples have been determined by these broad-band recoupling techniques, because the distances cannot be accurately deter-mined, owing to the contribution of dipolar couplings from neighboring spins.

Heteronuclear Distance MeasurementsRotational-echo double resonance (REDOR) NMR spectroscopy (37) is a pow-erful method for measuring weak heteronuclear dipolar couplings between un-like spins (labeled I and S) such as13C,15N, and31P. REDOR utilizes RF pulsesat the resonance frequency for the I or S spins to interfere with the effect ofMAS on the dipolar interactions. REDOR uses rotational echoes generated bysample spinning. A series ofπ pulses are applied to the I or S spins to preventthe dipolar IS coupling from refocusing at the time of the rotational echoes.Usually twoπ pulses are required in each rotational period of the magic anglespinning to ensure that the sign of the heteronuclear dipolar coupling is the sameat the beginning of each rotational period, so that the resulting phase angle canbe accumulated from one sample revolution to the next. The resonance inten-sities of the I spin are then monitored as a function of the rotor cycles with andwithout irradiation at the resonance of the S spin. The difference between twointensities depends exclusively on the heteronuclear dipolar coupling, yieldingthe internuclear distance between the two spins.

REDOR has several attractive features. Different from homonuclear distancemeasurements, REDOR relies only on the dipolar coupling between two spinsand requires no knowledge of the chemical shift tensors of the spins and theirrelative orientation, and therefore the data analysis for extracting dipolar dis-tances from REDOR experiments becomes simple. REDOR is also capable ofmeasuring relatively long distances, up to 10A, depending on the labeled spinpairs being studied, e.g.13C-15N, 13N-31P, or 13C-31P. REDOR spectroscopyis versatile and has been modified extensively for various systems. For ex-ample, transferred-echo double-resonance (TEDOR) NMR spectroscopy (43)utilizes coherence transfer between two coupled spins to eliminate unwanted

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background signals from uncoupled spins. Rotational-echo, adiabatic-passage,double resonance (REAPDOR) (35), derived from the combination of theprinciples for REDOR with that for transfer-of-populations-double-resonance(TRAPDOR) (33), is capable of measuring the distance between spin-1/2 andquadrupolar nuclei such as13C-14N, 13C-2H, and13C-23Na pairs. These tech-niques have great potential for studies in biological systems, including polypep-tides and membrane and structural proteins. For instance, nitrogen occurs inevery backbone structural element of proteins and 99.63% of naturally abundantnitrogen is14N; thus it would be convenient to utilize this spin in distance mea-surements, thereby avoiding isotopic labeling with two different nuclei. Manyproteins bind cations, and REAPDOR provides an important technique thatcould be used for measuring distances between the cation and various siteswithin the proteins.

REDOR and its related methods find their great utility in characterizing di-verse systems. Nishimura et al (76) determined the complete three-dimensionalstructure of the pentapeptide Leu-enkephalin dihydrate for the first time, on thebasis of six accurately measured interatomic13C/15N distances from REDORNMR spectroscopy, combined with some additional constraints from13C chem-ical shifts (54). Enkephalin is an endogenous morphine-like peptide, composedof two pentapeptides, Tyr-Gly-Gly-Phe-Met-OH (Met-enkephalin) and Tyr-Gly-Gly-Phe-Leu-OH (Leu-enkephalin). The sequence of the first four aminoacids is commonly found in the N-terminal region of endorphins and is responsi-ble for their biological activity through binding toδ-,µ-, orκ-receptors. For theLeu-enkephalin study, six samples were systematically synthesized with differ-ent pairs of13C/15N labels in the pentapeptide backbone (Figure 6), and then dis-tance constraints were measured between the13C/15N pairs. By using standardbond lengths and angles, these constraints were then converted topologicallyinto local torsion angles of constituent amino acid residues. This metric method(11) was described by Brenneman & Cross (4) for structural determination ofpolypeptides and was also used by others (27, 62, 30). It is important to notethat in order to derive accurate distance constraints, as discussed with homonu-clear constraints, when measuring long-range distances, dipolar contributionsfrom labeled nuclei of neighboring molecules must be considered. Nishimuraet al (76) found that the dilution effect onII (see Figure 6) is smaller than onI andIII and, thus, concluded that the peptides possibly form as an antiparallelβ-sheet structure such that one peptide interacts with two other peptides.

The REDOR experiments are usually implemented at low spinning speedsand over many rotor cycles so as to increase the size of the difference sig-nal, especially when the distance between two spins of interest is long ormolecular motions are present, resulting in weak dipolar couplings. Like manymultiple-pulse sequences, phase cycling (36, 67, 69) is employed in the REDOR

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254 FU & CROSS

Fig

ure

6Si

xpa

irs

of13

C/15

Nla

bels

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the

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.[R

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6).]

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experiments to partially compensate for errors due toπ pulse imperfections suchas inaccuracy of the flip angle and the resonance offset effect, but not for thefinite duration of the pulses. The REDOR experiments require analysis of thecomplete signal intensity to yield accurate distance information. The wholesignal intensity includes the centerband as well as all sidebands if the spinningspeed is not much larger than the size of the CSA for the spin under study. Itbecomes difficult to monitor the sideband intensities when the spinning is lowand the CSA is large, especially when sensitivity becomes a factor. The reasonfor this is that the dephasing of spinning sidebands strongly depends on therelative orientation of the chemical shift tensor and the internuclear vector (29).The dephasing behavior of individual sidebands may be different, so that anyindividual sideband dephasing does not represent the accurate distance. It isthus preferable to implement the REDOR experiments at high spinning speed inorder to concentrate the signal intensity into the centerband. High-field NMR isattractive because of greatly enhanced sensitivity as well as gains in resolution,but the CSA also increases, thereby increasing the demands on spinning speed.However, at very high spinning speed, REDOR tends to become inefficient dueto the fact that the finiteπ -pulse lengths correspond to a significant fraction ofthe rotational period (73, 103).

Recently, simultaneous frequency and amplitude modulation (SFAM) NMRspectroscopy (26) was proposed as an alternative to measuring internuclear dis-tances. For this experiment, the carrier frequency of one of the spins (e.g.15N)is modulated cosinusoidally while its RF amplitude is modulated as a sine wave.Different from REDOR, SFAM introduces a time dependence via modulationsto interfere with sample spinning and thus prevents the heteronuclear dipolarcouplings from being averaged out by sample spinning. Unlike the REDORexperiments, SFAM has no limitation on the strength of irradiation and thuscan be used at high field where high spinning speed is desirable. SFAM hasbeen proven to be very efficient over a broad range of spinning speeds and to beinsensitive to the RF inhomogeneity and resonance offset due to the fact that thecarrier frequency and the RF amplitude are modulated simultaneously (26, 51).

SFAM has great potential for measuring extremely weak residual dipolarcouplings. An exclusive example of combining distance constraints with ori-entational constraints was demonstrated for characterization of gramicidin (9).The structure of gA in membranes has been derived using orientation-dependentsolid-state NMR studies (55, 56), featuring an amino terminal to amino termi-nal hydrogen bonded single-stranded dimer with each monomer folding into aright-handedβ-helix of 6.5 residues per turn. However, it is also well acceptedthat the left-handed antiparallel double-stranded structure adopted by gA in ben-zene/ethanol, used to cosolubilize peptide and lipid prior to bilayer formation,converts readily to the channel state when inserted into the bilayer. The four

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256 FU & CROSS

tryptophan side chains may play an important role in the conversion process(M Cotten & TA Cross, unpublished results). Gramicidin M (gM), in which allTrp is replaced by Phe, is being used as a model to gain insight into the structuralconversion for which it is speculated that gA is inserted as a double-strandeddimer and then unscrews to form a single-stranded channel rather than insertedas monomers that dock to form the channel. Previous studies (8) indicated thatgM features a left-handed double-stranded dimer structure in lipid bilayers.Distance measurements permit determination of whether the dimer is a parallelor antiparallel structure. As illustrated in Figure 7a, the amide nitrogen of Phe11and the carbonyl carbon of Ala5 in the same monomer, specifically labeled, are10 A apart for both models, giving too small a13C/15N dipolar coupling to bedetected. The intermonomer distances between the labeled sites are 4 and 9Afor antiparallel and parallel models, respectively. For the former model, thedimer structure results in a reasonable13C/15N dipolar coupling, but not for thelatter one, even though the dimer undergoes a fast global rotation around itshelical axis, partially averaging the13C/15N dipolar coupling. The dephasing ofthe13C signal at 173 ppm indicates a residual dipolar coupling of 19± 3 Hz.Taking into account the orientation of the internuclear13C/15N vector with re-spect to the global motion axis (84), the distance between the13C/15N pair is4.5 ± 0.3A, consistent only with the antiparallel dimer structure.

TORSIONAL CONSTRAINTS

Although the measurement of relative tensor orientations was pointed out in1980 (68), until recently torsional constraints from solid-state NMR charac-terizing protein structures had received only limited attention. Torsional ordihedral angles define the relative orientations of rigid structure elements suchas an indole group, a methylene, or a peptide plane. Tensors are usually welldefined or can be independently characterized with respect to their molecularframe. For example, the CSA of the backbone carbonyl carbon is reasonablywell characterized, and the dipolar tensors are believed to be axially symmetricwith respect to internuclear vectors. Thus, a correlation between two tensors onadjacent structural elements leads to torsional constraints between these adja-cent elements. As discussed above, torsional information is indirectly availablefrom R2 (66) and REDOR experiments (29). However, the data analysis forextracting the torsional constraints is not staightforward; it depends on param-eters such as T2

ZQ in R2 experiments. Two-dimensional (2D) exchange NMRspectroscopy (53) that correlates two NMR frequencies governed by selectedinteractions of interest in different time domains provides a direct approach forobtaining torsional constraints. However, the earlier 2D static exchange NMRspectroscopy finds limited applications because of low sensitivity and poor

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Figure 7 (a) Two dimer structures of gramicidin M in hydrated dimyristoylphosphatidylcholine(DMPC) bilayers. (Solid circles) The labeled sites of13C and15N. (b) A set of13C CPMAS spectra atdifferent dephasing times with (left) and without (right) the simultaneous frequency and amplitudemodulation (SFAM) irradiation on the15N channel. The spectra were recorded at 315◦K (abovethe phase transition temperature of DMPC). The peak at 174 ppm is not affected by dephasingand is therefore assigned to the carbonyl group of the lipids. The peptide resonance at 173 ppm isattenuated by the SFAM irradiation.

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258 FU & CROSS

resolution associated with broad powder pattern line shapes. This is particularlya problem for complex systems such as peptides. MAS enhances sensitivity andimproves resolution, but it also suppresses informative anisotropic interactions,including orientations with respect to the molecular frame.

Recent developments for measuring torsional constraints have focused mainlyon how to avoid having informative anisotropies averaged by MAS. Significantprogress has been made toward developing a powerful and attractive way todirectly characterize local conformations of polypeptides and proteins. Asdemonstrated by Costa et al (6), direct measurement of torsion angles clari-fied that the Gly37-Gly38 amide bond in theβ-amyloid fibril was in thetransconfiguration, which was ambiguous with just the distance constraints. Rotor-synchronized exchange spectroscopy (3) originally developed for studies ofslow molecular motion and chemical exchange has been utilized in structuralstudies (105, 109). Low spinning speeds are required to generate spinning side-bands that contain chemical shift anisotropic information. With careful timingof pulses in synchrony with sample spinning, the chemical shift tensors of twospins cross-talk during the mixing time, developed by proton-driven13C-13Cspin diffusion, resulting in intersite cross peaks that contain orientational in-formation between two chemical shift tensors. The torsional constraints canthus be extracted with knowledge of the orientations of the two tensors in theirrespective local molecular frame. The measurement on the13C doubly labeledAla1 and Gly2 of a model tripeptide, AGG, led to backbone torsion angles ofφ,ψ = −78◦,168◦, in excellent agreement withφ,ψ = −83◦,170◦ determinedby neutron diffraction (105, 109).

Ishii and coworkers (49, 52) incorporated recoupling techniques under MASwith traditional static 2D exchange spectroscopy. The powder patterns forinteractions of interest are restored in different time domains connected by amixing time, during which the exchange mechanism is chosen. For instance, inan O-C1-Cα-H system, the CSA of C1 is restored by the 6π -pulse sequence (104)in the t1domain, and the magnetization exchange between C1and Cα is driven bythe R2TR (52). Finally, the Cα signal is selected and evolved under Cα-H dipolarcoupling in the t2 domain, leading to a CSA-DD 2D correlation spectrum. Thisleads to characterization of theψ torsion angle. For an H-15N-13C-H system,powder patterns of15N-H and13C-H dipolar couplings are restored in the t1and t2 domains that are connected by cross polarization between13C and15N,resulting in a DD(NH)-DD(CH) correlation (49) and leading to characterizationof the φ torsion angle. Hong et al (45, 46) proposed a 2D dipolar-chemicalshift (DIPSHIFT) experiment in which dipolar sidebands composed by13C-Hand15N-H dipolar couplings are present in the t1 domain, whereas the high-resolution13C or15N chemical shift evolves in the t2 domain. Because the13C-Hand15N-H dipolar tensors are known to be axially symmetric with respect to

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their internuclear vector, torsional constraints can be accurately extracted from2D DD-DD or DIPSHIFT correlation spectra without major assumptions.

Particularly promising for determination of torsion angles in complex sys-tems such as peptides is 2D double-quantum (DQ) NMR spectroscopy (91).DQ is typically used as a spectral filter to eliminate the unwanted signals fromuncoupled spins, leaving only coupled spin pairs of interest, thus simplifyingthe observed spectra. The ability to efficiently generate DQ coherence underMAS (32, 65) has led to diverse applications of 2D DQ NMR spectroscopy inpeptides and proteins, with the advantages of high sensitivity and high reso-lution. Figure 8 demonstrates the advantages of DQ filtration. Schmidt-Rohr(92) demonstrated torsion angle determination on static samples. In the exper-iment, DQ coherence was generated and evolved in the t1 domain under13C-Hdipolar couplings. This dipolar modulated DQ coherence was converted tosingle-quantum coherence for detection under13C chemical shift and13C-13Cdipolar coupling. Similarly, Gregory et al (30) generated DQ coherence underMAS conditions by homonuclear dipolar recoupling using a windowless multi-pulse irradiation technique (DRAWS) (31) and leading to determinations of themutual orientation of two or more CSA tensors with high accuracy, assumingthat these tensors are well characterized with respect to the molecular frame.Levitt and coworkers used the C7 sequence (65) to efficiently generate andreconvert DQ coherence (22), independent of chemical shift tensors. The DQcoherence is evolved under13C-H dipolar couplings in a H-13C-13C-H system inthe t1 domain, and strongly depends on the orientation of the two13C-H dipolarcouplings, leading to extraction of torsional constraints without knowledge ofchemical shift tensors (21, 23, 24).

FUTURE DIRECTIONS

The potential for solid-state NMR to become a major structural technique isbeginning to be realized. However, the most critical hurdle in NMR remainssensitivity. Although sensitivity has improved by nearly 1000-fold over thepast three decades, another order of magnitude would tremendously facilitatethese experiments. Signal averaging time would be greatly reduced, sample sizecould be further reduced, and signal-to-noise ratios could be improved. NMRtechnology is on the brink of great advances: The first commercial 900-MHzinstruments will be delivered in 1999, the first prototype high-temperature su-perconducting probes have been delivered, and cryocooled probes are on thehorizon. Because signal-averaging time decreases in proportion to B3

o, a givensignal-to-noise ratio achieved in 2 days at 600 MHz could be achieved in 14 hon a 900-MHz spectrometer. Even higher fields are possible (10, 77). Resis-tive magnets at the National High Magnetic Field Laboratory are being used

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for NMR spectroscopy (Figure 9). At this time, resistive magnets operateat fields up to 33 T (1.4 GHz for1H) and a 25-T, 52-mm bore magnet hasbeen shown to be temporally stable to 1 ppm, and 1-ppm homogeneity over aspherical volume with a 1-cm diameter is anticipated by the end of 1998. High-temperature superconducting and cryocooled probes generate substantial sen-sitivity improvement for solution NMR (factors of 4 or 5), but the technologyhas yet to be developed for solid-state NMR. Polarization transfer methodssuch as dynamic nuclear polarization have demonstrated factors of 200 im-provement in sensitivity on model systems (38). These advances in technologywill greatly benefit structure determination by solid-state NMR.

Until recently, few membrane proteins had been cloned, overexpressed, andpurified in sufficient quantity (5–10 mg) for NMR spectroscopy. However, thisis rapidly changing, and new approaches for splicing protein domains (112)suggest that uniform labeling of domains within proteins rather than uniformlabeling of the entire protein will soon be possible. This will improve spectralresolution and spectral interpretation.

The PISEMA experiments (110) have greatly improved the resolution ofstatic solid-state spin interaction correlation spectra and have been successfullyused to obtain orientational constraints from uniformly labeled proteins. Incontrast, limited success has been achieved by the use of distance constraintsand torsional constraints from unoriented uniformly labeled samples. A spinpair of interest needs to be isolated from the spin network so that the effect ofneighboring spins in the network can be eliminated, providing accurate distanceconstraints and torsional constraints. A new methodology like QUIET NOESY(113) in solution NMR has yet to be developed for effectively quenching unde-sirable contributions and thus allowing for accurate measurements of distanceconstraints and torsional constraints in uniformly labeled samples.

The technology for structural characterization is also rapidly advancing.Many research groups have started to work in this area, developing new meth-ods from magic angle spinning of oriented samples (28), to spin diffusion asa measure of depth in lipid bilayers (61), to a one-dimensional heteronuclearexperiment (63). The utilization of more than one type of constraint is justbeginning to happen (6, 9, 18, 30), and an efficient algorithm for solving struc-tures has only just begun to be developed (4, 57). Yet a high-resolution structureof a peptide in a lamellar lipid phase has been solved where crystallographic

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 8 (a) Molecular structure of the retinylidene chromophore in rhodopsin. (Asterisk) 13Clabels. (b) 13C CPMAS spectrum of membrane-bound bovine rhodopsin. (c) Double-quantumfiltered13C CPMAS spectrum using the C7 pulse sequence. [Reprinted with permission from Fenget al (21).]

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Figure 9 2H nuclear magnetic resonance spectra of d4-Ala5–labeled gramicidin A in oriented lipidbilayers aligned with the bilayer normal parallel to the magnetic field axis. The high-field spectrumwas obtained in a resistive magnet operating at 13 MW and temporally stabilized to 1 ppm. The fieldinhomogeneity was significantly worse than in the 14.1-T superconducting magnet contributing tothe line width of these resonances. Despite this, excellent orientational constraints are obtained ona much smaller sample than was used for the 14.1-T spectrum. [Reprinted with permission fromCotten et al (10).]

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efforts have been unsuccessful; high-resolution spectra of a 200-amino acid,uniformly 15N-labeled protein in a lipid bilayer has been observed; the structureof silk fibroin has been refined and the structure of aβ-amyloid peptide char-acterized. These document that the potential for solid-state NMR is becominga reality, and further advances in the technology will only enhance the generalapplicability of solid-state NMR (a) to a broad range of proteins that have notbeen readily approachable by other structural methods and (b) to enhance theefficiency with which structures can be determined.

ACKNOWLEDGMENTS

TAC gratefully acknowledges support from the National Institutes of Health(R01 AI-23007) and the National Science Foundation (MCB-9603935). A por-tion of the work reported in this review was performed at the National HighMagnetic Field Laboratory, supported by NSF Cooperative Agreement DMR-9527035 and the State of Florida.

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Annual Review of Biophysics and Biomolecular Structure Volume 28, 1999

CONTENTSRAMAN SPECTROSCOPY OF PROTEIN AND NUCLEIC ACID ASSEMBLIES, George J. Thomas Jr. 1

MULTIPROTEIN-DNA COMPLEXES IN TRANSCRIPTIONAL REGULATION, Cynthia Wolberger 29

RNA FOLDS: Insights from Recent Crystal Structures, Adrian R. Ferré-D'Amaré, Jennifer A. Doudna 57

MODERN APPLICATIONS OF ANALYTICAL ULTRACENTRIFUGATION, T. M. Laue, W. F. Stafford III 75

DNA REPAIR MECHANISMS FOR THE RECOGNITION AND REMOVAL OF DAMAGED DNA BASES, Clifford D. Mol, Sudip S. Parikh, Christopher D. Putnam, Terence P. Lo, John A. Tainer 101

NITROXIDE SPIN-SPIN INTERACTIONS: Applications to Protein Structure and Dynamics, Eric J. Hustedt, Albert H. Beth 129

MOLECULAR DYNAMICS SIMULATIONS OF BIOMOLECULES: Long-Range Electrostatic Effects, Celeste Sagui, Thomas A. Darden 155

THE LYSOSOMAL CYSTEINE PROTEASES, Mary E. McGrath 181

ROTATIONAL COUPLING IN THE F0F1 ATP SYNTHASE, Robert K. Nakamoto, Christian J. Ketchum, Marwan K. Al-Shawi 205

SOLID-STATE NUCLEAR MAGNETIC RESONANCE INVESTIGATION OF PROTEIN AND POLYPEPTIDE STRUCTURE, Riqiang Fu, Timothy A. Cross 235

STRUCTURE AND CONFORMATION OF COMPLEX CARBOHYDRATES OF GLYCOPROTEINS, GLYCOLIPIDS, AND BACTERIAL POLYSACCHARIDES, C. Allen Bush, Manuel Martin-Pastor, Anne Imbery 269

THE PROTEASOME, Matthias Bochtler, Lars Ditzel, Michael Groll, Claudia Hartmann, Robert Huber 295MEMBRANE PROTEIN FOLDING AND STABILITY: Physical Principles, Stephen H. White, William C. Wimley 319

CLOSING IN ON BACTERIORHODOPSIN: Progress in Understanding the Molecule, Ulrich Haupts, Jörg Tittor, Dieter Oesterhelt 367

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