Lipid-peptide interaction investigated by NMR

CHAPTER 6 Lipid-Peptide Interaction Investigated by NMR Kiaus Gawrisch* and Bernd W. Koenigt *Laboratory of Membrane Biochemistry and Biophysics, NIAAA, National Institutes of Health, Rockville, Maryland 20852; tIBI-2: Institute of Structural Biology, Research Center Jtilich, D-52425 Jiilich, Germany

I. Introduction II. Peptide Structure in the Membrane-Bound State A. Peptide Structure in Membrane-Mimetic Environments Studied by High-Resolution NMR B. Membrane-Bound Structure of Weakly Interacting Peptides C. Solid-State NMR Approaches to Studying Peptide Structure in the Membrane-Bound State III. Structure and Dynamics of the Lipid Matrix A. 31p Anisotropy of Chemical Shift B. 2H NMR C. Magic Angle Spinning NMR D. Influence of Peptide Binding on Lipid Structure IV. Future Directions References

Nuclear magnetic resonance (NMR) enables the investigation of peptides as well as the lipid matrix to which the peptides bind. Peptide structure is studied by both high-resolution and solid-state NMR methods. Application of high-resolution NMR requires rapid isotropic peptide motions, achieved by conducting experiments in membrane-mimetic environments, that is, organic solvents or detergent solutions. Solid-state NMR investigations are directly conducted on peptides in the membrane-bound state, but experiments often require elimination of peptide motions by freezing or dehydrating samples. Novel NMR techniques utilizing rapid magic angle spinning or oriented peptide/lipid samples have dramatically

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improved resolution of solid-state NMR experiments in recent years. Peptide binding to lipids changes the membrane hydrophobic thickness and area per molecule, both reflected in lipid order parameter changes. Nuclear Overhauser enhancement spectroscopy (NOESY), in combination with magic angle spinning, is a novel approach to studying structure and dynamics of the lipid matrix. The experiments provide deeper insight into the tremendous conformational and spatial disorder of lipids in biomembranes.

i. INTRODLICTION High-resolution NMR is a reliable tool for structural investigations of peptides and small proteins in solution. The molecular weight limit for complete resolution of structure is near 30,000 daltons. Under favorable circumstances, this range can be extended to values near 100,000 daltons using transverse relaxation-optimized spectroscopy (TROSY) sequences (Pervushin et al., 1997) in combination with high magnetic field strength and extensive isotopic labeling of the protein. The complexes of peptides with micelles fall within the permissible weight range and can be studied by proven high-resolution NMR approaches. Furthermore, peptide structures similar to those of the membrane-bound state can be investigated on dissolved peptides under solvent conditions that simulate a membrane environment. The aggregates of peptides with lipid bilayers have molecular weights that far exceed the limits of high-resolution NMR. Even the complexes between peptides and small sonicated liposomes, with diameters of less than 1000 A, tumble too slowly for high-resolution NMR applications. Solid-state NMR is used to study the structure of peptides that are bound to membranes. The method takes advantage of the spatially anisotropic interactions that shift, split, and broaden NMR resonance lines. Conformation and dynamics of lipids and changes in lipid packing as a result of peptide binding can be studied as well. The term "structure" in the context of peptide-lipid interaction must be used with great caution. Models that show lipids and peptides in static arrangements have shaped our perception. In reality, the lipids in the matrix of biomembranes are liquid-crystalline and exchange rapidly among an infinite number of conformations. Peptides may have rigid backbones, but can also retain significant degrees of flexibility. Lifetimes of noncovalent interactions between peptides and lipids range from pico- to milliseconds. The lipid matrix easily adjusts to the needs of peptide-lipid interaction. Average lipid conformation and lipid lateral organization may change as a result of interactions with peptides. Although NMR competes with other methods for the structural investigation of peptides, it is the method of choice for studying the dynamics of lipid-peptide complexes. Motional correlation

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times that cover 12 orders of magnitude, from picoseconds to seconds, are accessible by NMR. The goal of this chapter is to provide general knowledge about opportunities and limitations of NMR in the study of peptide-lipid interactions. Technical details that would appeal mostly to NMR spectroscopists have been avoided, but can be found in the cited literature. Because of the authors' involvement in studies on the interaction of amphipathic peptides with lipid/water interfaces, most of the experimental examples are taken from this field of research.

II. PEPTIDE STRUCTURE IN THE MEMBRANE-BOUND STATE

A. Peptide Structure in Membrane-Mimetic Environments Studied by High-Resolution NMR The majority of short peptides in aqueous solution are very flexible. Peptide binding to a lipid membrane often results in the formation of secondary structure or even of a unique peptide conformation. The structural transition reflects the change in the microenvironment of the peptide upon membrane binding, that is, the transfer down a steep polarity gradient from the very polar aqueous phase to the complex environment of a hydrophobic/hydrophilic interface. Organic solvents of low polarity or detergent micelles in water may promote the formation of peptide structures similar to what is found in peptide-membrane complexes. Studies that mimic membrane conditions are frequently referred to as studies in membrane-mimetic environments. For example, methanol or trifluoroethanol/water mixtures facilitate formation of a helices, provided the peptide has helical propensity. The environment of detergent micelles is a better choice for structural studies on membrane-spanning segments. In addition, micelles are also an appropriate medium for amphipathic peptides that require a polar and/or a charged interface region for proper folding. The NH--NH region of a two-dimensional (2D) 1H NOESY spectrum ofa 21-residue amphipathic peptide from the envelope glycoprotein gp41 in sodium dodecylsulfate (SDS) solution is presented in Fig. 1. The choice of experimental conditions is of fundamental importance. The peptide structure may depend on solvent properties (Gesell et al., 1997) as well as pH, ionic strength, and temperature. When detergent micelles are used, detergent concentration is particularly important to ensure both a unique peptide structure as well as a proper aggregate size to enable detection of well-resolved NMR spectra (McDonnell and Opella, 1993). Sodium dodecylsulfate and dodecylphosphocholine (DPC) micelles are most frequently used to mimic negatively charged and zwitterionic membranes, respectively. Experiments may benefit from the use

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ppm FIGURE 1 High-resolution NMR study of a cytolytic peptide fragment, 828-848 (P828), from the carboxy terminus of the envelope glycoprotein gp41 of HIV-1 in SDS micellar solution. Shown is the amide region of the IH 2D NOESY NMR spectrum of 5.3 mM P828 in 277 mM 1-stearoyl-d352-oleolyl-sn-glycero-3-phosphocholinesolution at T = 60°C. Sequential NH-to-NH crosspeaks are labeled. Figure reproduced from Koenig et al. (1995).

of perdeuterated detergent molecules to prevent signal superposition with the much weaker peptide signals. Equivalence of peptide structure in the chosen membranemimetic environment with the membrane-bound structure must be demonstrated. This is conveniently done by methods, like circular dichroism (CD), which can be applied to solutions of peptide interacting with small unilamellar liposomes as well as to peptides in organic solvent or in micelle solution. Matching of the CD spectra of a peptide in a membrane-mimetic environment and in a real membrane strongly suggests similar peptide conformations (Gawrisch e t al., 1993; Koenig e t al., 1995).

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The rationale behind the use of membrane-mimetic environments is their compatibility with high-resolution NMR experiments for peptide structural investigations. Peptides in organic solvents or micelles perform rapid, isotropic rotational diffusion. The rotational correlation time of a micelle-bound peptide depends on the size of aggregates and the viscosity of the medium. Micelle size is critical: A small peptide with a weight of 2000 daltons bound to a micelle may behave spectroscopically like a protein in water with a weight of 20,000-30,000 daltons. At such molecular weights, NMR spectra may suffer from line broadening and short spin-spin relaxation times, and NOESY cross-relaxation may be under the influence of spin diffusion. Structural determination of peptides adopting a unique conformation in membrane-mimetic environment follows the same strategy as NMR structure determination on soluble proteins (Wtithrich, 1986). Two-dimensional 1H-1H experiments [total correlation spectroscopy (TOCSY) or homonuclear HartmannHahn (HOHAHA) spectroscopy (Bax, 1989), correlation spectroscopy (COSY; Wtithrich, 1986), NOESY (Kumar et al., 1980), and rotating-frame nuclear Overh a u s e r e n h a n c e m e n t s p e c t r o s c o p y ( R O E S Y ; B o t h n e r - B y e t a l . , 1984)] on unlabeled peptide will usually provide sufficient resolution for complete resonance assignment of all peptide protons using the sequential resonance assignment technique (W~ithrich, 1986). In the case of severe proton signal overlap, the larger dispersion in the 15N and 13C dimensions is exploited to assign the protons that are directly bound to ISN and 13C nuclei, for example, by two-dimensional experiments with inverse detection of the insensitive nuclei via the resonance of protons, like heteronuclear single quantum coherence (HSQC) and heteronuclear multiple quantum coherence (HMQC) (Bax et al., 1989), or by three- and four-dimensional, heteronuclear-edited, proton-detected experiments (Clore and Gronenborn, 1991). At peptide concentrations in the millimolar range, the 13C signals can be detected at natural abundance (Koenig et al., 1999a). However, inverse experiments with 15N spins require isotope labeling. The chemical shift values of 1H (Wishart et al., 1991) and 13C spins (Spera and Bax, 1991; Wishart and Sykes, 1994) are site-specific indicators of the presence of secondary structure (see Fig. 2). Similarly, the 1H-1H nuclear Overhauser effect (NOE) connectivity pattern, which reflects spatial proximity of peptide protons, provides information on the local secondary structure (Wtithrich, 1986). The combination of strong NH/NH(i, i + 1) NOE crosspeaks with weak ctH/NH(i, i + 1) crosspeaks indicates or-helical structures (see Figs. 1 and 3), whereas the combination of strong ctH/NH and weak NH/NH NOE crosspeaks is expected for r-sheet structures. In addition to approximate interproton distance constraints from NOESY or ROESY spectra, torsion angles (see Fig. 4) in rigid peptides can be restrained. T h e 3JNH_Hceand 3 JHo~-H3 scalar coupling constants are related to protein ~p and X1 torsion angles via empirical Karplus relations

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FIGURE 2 The peptide P828 is not structured in water, but converts to a partially helical conformation in SDS micelles. The structural transition is reflected in characteristic changes in backbone chemical shifts. (a) Conformation-induced ~ secondary shift. Shown is the difference between the chemical shift values observed in water and in SDS solution. (b) Digital values representing the 13Ca secondary shifts of P828S in SDS micelles. Depending on the deviation of the chemical shift from average values, a digital value of 1, 0, or -1 is assigned to every Ca carbon. The digital value + I for amino acids from valine V2 to arginine R14confirmsthe existenceof a helical conformation in this region of the molecule. Figure reproduced from Koenig et al. (1999a).

(Pardi et aL, 1984). They are obtained from IH-15N H M Q C (Forman-Kay et al., 1990; Kay and Bax, 1990) and homonuclear primitive exclusive C O S Y (PE.COSY; MUller, 1987) correlation spectra, respectively. Torsion angles ~p have been determined from cross-correlated relaxation rates (Reif et al., 1997; Sprangers et al., 20O0). The NMR-derived geometric constraints are inserted as interaction potentials into molecular dynamics calculations (Nilges, 1996), which provide sets o f lowenergy peptide structures. Alternatively, peptide conformation can be evaluated by distance geometry calculations based on N M R distance constraints (Okada et al., 1994). In a membrane-mimetic environment, and also when bound to membranes, small peptides often retain a significant degree of flexibility rather than form a rigid structure. Fluctuations in the m i c e l l e - w a t e r interface are significantly larger than structural fluctuations in the lipid/water interface. Therefore it is likely that peptides in micelles show increased conformational flexibility. When peptides are flexible, spectroscopic parameters reflect the averaging over the entire set of

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FIGURE 3 Summaryof 1H-1HNOE connectivitiesof peptide P828S, which enable identification of secondary structure. The index i, is the number of the amino acid in the peptide sequence. For the crosspeaks between neighboring amino acids, NH(i)/NH(i + 1), and for H(i)/NH(i + 1), the height of the boxes is proportional to the measured NOE intensities. Question marks indicate crosspeaks that cannot be assessed due to signal overlap. Medium-range NOE interactions over three and four amino acids that are characteristicof helices are summarizedin the four lower rows. Solid bars indicate unique crosspeaks, and dotted lines are used for connectivities whose presence or absence cannot be assessed,due to spectraloverlap.Observationof medium-rangecrosspeaksof the peptideregion near the C-terminuswas not possible due to the proximity of the correspondingH resonancesto water.According to induced changes in chemical shift, this region is unstructured. Figure reproduced from Koenig et al. (1999a).

peptide structures. Quantitative analysis of spin-lattice and spin-spin relaxation data provides a more detailed description of the dynamics of conformational transition (Ishima and Torchia, 2000). The amplitude of the heteronuclear ]SN-]H nuclear Overhauser enhancement is very sensitive to motions with frequencies near 109 Hz, which allows a qualitative distinction between mobile and rigid sites in a peptide (Bogusky et al., 1987, 1988). The relative intensities of sequential N H / N H and (~H/NH crosspeaks in homonuclear N O E S Y spectra reveal conformational averaging (Dyson and Wright, 1991; Koenig et al., 1999a). The temperature dependence o f the chemical shift of peptide amide protons is sensitive to the formation of hydrogen bonds. A linear dependence with small temperature coefficients between 0 and - 3 p p b / K indicates regular secondary structure elements stabilized by intramolecular hydrogen bonds, whereas values between - 6 and - 1 0 p p b / K are typical of hydrogen bonding between peptide and solvent (Deslauriers and Smith, 1980). Intermediate values m a y reflect conformational flexibility (Koenig et al., 1999a).

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OH FIGURE 4 Standard nomenclature for atoms and torsion angles in a peptide chain. A torsion angle defines the amount of twist about a bond axis and is defined, for a bonded series of four atoms (A--B--C--D), as the angle of rotation about bond B--C required to make the projection of bond axis B--A coincide with the projection of bond axis C--D, when viewed along the B--C direction. The angle is positive for clockwise rotation. Backbone torsion angles 4, that is, (C~_l--Ni--ot Ci--C~ ), and ~, that is, (Ni---otCi--C~--Ni+l) , and side-chain torsion angle ;~l, that is, ( N i - - o t C i - - f l C i - - y C i ) , are particularly useful.

B, Membrane-Bound Structure of Weakly Interacting Peptides Resonance signals of peptides that interact weakly with the surface of liposomes, but spend most of their time in solution, remain highly resolved. However, the membrane-peptide interactions cause significant NMR relaxation enhancement. The NOE cross-relaxation rates depend on the motional correlation time rc of the internuclear vector pairs of interacting protons. The nuclear Overhauser effect is positive for short re and negative for long ~c, because of differences in the correlation time dependence of relaxation pathways (Neuhaus and Williamson, 1989). At the field strength of modern high-field NMR spectrometers, the crossover in the sign of NOE, where crosspeaks are very weak or undetectable, occurs at rc between 10 -9 and 10 -l° s. These are typical correlation times of short peptides with about 10 amino acids in aqueous solution. Cross-relaxation of peptides that briefly bind to membranes is dominated by the much longer correlation times of slowly tumbling peptide/lipid complexes in the membrane-bound state. The proton distance information of the membranebound state is "transferred" to the solution-state NMR signals. Therefore, such experiments are referred to as transferred NOE (TrNOE) measurements (Clore and Gronenborn, 1982, 1983; Ni, 1994). Observation of TrNOEs requires that the peptide off-rate is fast relative to both the longitudinal relaxation rate of the bound peptide and the inverse of the NOE mixing time. It is often necessary to lower the natural affinity of the peptide for membrane interaction to meet the

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stringent off-rate requirements. This may be achieved by modifying the lipid composition (Z. Wang et al., 1993), varying the pH or ionic strength of the solution (Anglister and Zilber, 1990; Koenig et al., 1999b), or inducing conservative mutations in the peptide sequence (Anglister and Zilber, 1990; Koenig et al., 2000). Higher temperature also enhances the off-rate (Koenig et al., 2000). It may be advantageous to record NOESY spectra of the peptide both in the presence and the absence of membranes and to subtract the NOE of peptide acquired without the presence of membranes to remove NOE contributions from the free peptide. The TrNOE method has been successfully used to study the membrane-bound conformation of peptides such as mastoparan-X (Wakamatsu et al., 1992), melittin (Okada et al., 1994), neuropeptides (Bersch et al., 1993), and signal peptides (Z. Wang et al., 1993), as well as the structure of peptides bound to antibody fragments (Anglister and Zilber, 1990). It was observed that very low tumbling rates of peptides in the membrane-bound state make TrNOE experiments prone to spin diffusion, potentially resulting in crosspeaks between protons farther apart in space than the longest measurable NOE distances of 5 ]~. Great care must be taken to properly design and interpret TrNOE experiments in order to avoid or at least to recognize such artifacts (Arepalli et al., 1995; Ni, 1994).

C. Solid-State N M R A p p r o a c h e s to Studying Peptide Structure in the Membrane-Bound State

Permanent attachment of peptides to membranes or incorporation into membranes results in an anisotropy of peptide orientation that is not averaged out by fast motions. Solid-state NMR is the method of choice for investigating long-lived peptide-lipid complexes. It allows characterizing the structure of peptides that are immobilized on the relevant NMR time scale. There is no need to use membranemimetic environments. Many solid-state NMR methods work best with frozen or dehydrated samples, where most peptide motions are suppressed. Unfortunately, freezing and dehydration can alter the structure and dynamics of peptidemembrane complexes. Anisotropic interactions between magnetic nuclei and their environment dominate the appearance of solid-state NMR spectra. They result in orientationdependent shifts and splittings of peptide resonances. The magnitude of these effects is very sensitive to the orientation of bond vectors relative to the external magnetic field and to the distance between interacting nuclei. NMR parameters of interest include the 15N-chemical shift anisotropy (CSA) of peptide amides, the 13C-CSA of peptide carbonyl and Ca carbons, dipolar couplings between 1H, ~SN, and 13C spins of the peptide backbone, and quadrupolar splittings of 2H nuclei attached to amide nitrogens, Ca carbons, or side-chain carbons.

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There is a tradeoff between the ability to study peptides in the membrane-bound state and spectral resolution. Anisotropic interactions in solid-state NMR severely broaden resonance lines, resulting in low sensitivity and signal overlap. In recent years these limitations have been partially overcome by new approaches, for example, by using uniaxially oriented membranes or by applying magic angle spinning (MAS), which eliminates excessive signal broadening from anisotropic interactions. Although structural studies on large membrane proteins are still challenging, investigation of membrane-bound peptides with these techniques is very rewarding.

1. Angular Dependence of Interactions a. C h e m i c a l Shift. The chemical shift interaction of a nuclear spin is a tensor quantity. The experimentally observed chemical shift value is a function of both the magnitude of principal tensor components and their orientation relative to the direction of the external magnetic field Bo. Orientation of tensor axes relative to the molecular frame of peptides is determined experimentally or by quantum chemical calculations. Amide 15N and carbonyl 13C chemical shifts contain valuable information on the orientation of the peptide plane relative to the Bo field. In general, the amide 15N chemical shift tensor (~r33 > cr2: >_ O'll ) in peptide bonds is almost axially symmetric (a22 ~ cr11), with an asymmetry Act = cr33 - 1/2(cr22 + ~rl1 ) ~'~ 150 ppm (Harbison et al., 1984; Oas et al., 1987a; Wu et al., 1995). The principal axis, a33, is oriented close to the peptide plane and forms an angle of about 15-20 ° with the N--H bond axis (Brender et al., 2001). The dispersion of the isotropic amide 15N chemical shift in proteins is small compared to A~r and amounts to "-~20 ppm (Brender et al., 2001). Therefore, the orientation of an immobilized N--H bond vector relative to the external magnetic field can be estimated from a single amide 15N resonance frequency measurement. For example, amide 15N frequencies of peptides immobilized in uniaxially aligned membranes have been used to distinguish transmembrane and membrane-parallel orientations of helical segments (Bechinger et al., 1991, 1996). Membrane alignment with low mosaic spread is critical in such experiments and may be accomplished by spreading the membranes between stacked glass slides (McDonnell et al., 1993). Samples are oriented with the membrane normal parallel to the B0 field of the NMR magnet, using fiat-coil probes (Bechinger and Opella, 1991). The N--H bond in ot helices is, in first approximation, oriented parallel to the helix axis (Shonet al., 1991). Amide 15N resonance frequencies close to 220 and 80 ppm (relative to 15NH3) are expected for the transmembrane and surface-parallel orientations of a helices, respectively. Alternatively, alignment can be achieved by using magnetically oriented lipid bilayer fragments in solution (bicelles), allowing either parallel or perpendicular orientation of the membrane normal relative to B0 (Howard and Opella, 1996; Prosser et al., 1998; Sanders and Schwonek, 1992).

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Bicelle samples may allow for a higher filling factor of NMR coils, resulting in higher sensitivity. The 13C-CSA of the carbonyl carbon in peptide bonds can also be used to estimate the orientation of the peptide plane relative to the B0 field. Data show only moderate variation of the carbonyl 13C chemical shift interaction tensors in peptide bonds (Oas et al., 1987b; Separovic et al., 1990; Stark et al., 1983). The a l l and 0"22components are located in the peptide plane with 0-22 almost parallel to the C=O bond. The principal value and the orientation of the 0-22component are affected by hydrogen-bonding interactions (Asakawa et al., 1992). Analysis of the carbonyl 13C chemical shift has been used to study the orientation of gramicidin and melittin in uniaxially oriented membranes (R. Smith et al., 1989, 1994). b. 2HN M R Q u a d r u p o l a r Interactions. Orientation of 2H--N and 2H--C bonds with respect to the external magnetic field is derived from the strength of the quadrupolar interaction, which splits 2H NMR signals into a doublet. Typical static quadrupolar coupling constants are 150 kHz for 2H--N bonds (LoGrasso et al., 1988) and 168 kHz for 2H--C bonds (Burnett and Muller, 1971). In first approximation, the electric field gradients in 2H--N or 2H--C bonds, which act on the quadrupole moment of 2H nuclei, are axially symmetric around the bond axis. The angular dependence of quadrupolar interactions on the angle 0 between the direction of B0 and the chemical bond axis is given by a second-order Legendre polynomial function, P2 = [3 cos2(0) - 1]/2. This function changes sign at the magic angle of 54.7 °. Measurement of the sign of quadrupolar splittings is difficult and often omitted. This results in an additional degeneracy of bond orientation. Bond vector motions reduce quadrupolar splittings; the measured values reflect the dynamic average of bond vector orientation. Labile amide protons of peptides are easily deuterated by 2H exchange labeling (Datema et al., 1986). Signal superposition with other deuterated residues, such as hydroxyl groups, amino groups, and 2H20, as well as rapid back-exchange in H20 may complicate the NMR study. Peptide amide 2H--N bond orientation relative to the membrane normal is reflected in 2H NMR spectra of 2H-exchange-labeled peptides immobilized in uniaxially aligned membranes. The technique was used to discriminate among several structural models of gramicidin in membranes (Prosser et al., 1994). Specific deuterium labeling of nonlabile peptide hydrogens in combination with 2H NMR on gramicidin immobilized in oriented membranes was used to study the average orientation of 2H--Ca bonds (Hing et al., 1990), amino acid side chains (Killian et al., 1992; Lee et al., 1995), and tryptophane indole rings (Koeppe et al., 1994). Deuterium spectra of 2H-labeled peptides in nonoriented membranes report on the dynamics and on the local membrane environment of the labeled site (Dempsey et al., 1987; Koenig et al., 1999a). Recently, an approach that greatly enhances sensitivity and selectivity of detection of quadrupolar splittings was developed. The 2H NMR spectrum of a

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nonspinning solid was recorded indirectly through its modulation of the much more intense signal of a nearby 1H nucleus. A 15-fold enhancement of sensitivity was reported (S. Liu and K. Schmidt-Rohr, personal communication, 2001). Furthermore, this technique allows suppression of signals of highly mobile nuclei (e.g., the intense 2H20 signal). c. Dipolar Interactions. The strong distance dependence of the dipolar interaction can be used to determine distances within peptides. Nuclei with nonzero spin possess a magnetic moment, causing a dipole field, which modifies the effective field at the site of nearby nuclei. The strength of the dipole field Hij depends on the inverse third power of the internuclear distance ri~-3 and on the angle 0 between the internuclear vector and external B0 field: H~ = yi[3 COS2(0) -- 1]/r 3. The upper limit of accessible distances is 13 ,~ or less, depending on the gyromagnetic ratio YI of the interacting nuclei. Although measurement of dipolar interactions between nuclei in oriented samples is feasible, experiments are almost exclusively done under MAS conditions, which greatly improve the resolution and the intensity of resonance lines. Samples are packed into rotors that spin at rates in the kilohertz range with the rotor axis oriented under the magic angle, 0 = 54.7 °, relative to the external magnetic field. Fast rotation reduces the resonance linewidth by averaging anisotropic interactions. The strength of dipolar interactions can be measured with the following two techniques.

2. Rotational Resonance (RR) and Rotational-Echo Double Resonance (REDOR) In the RR experiment, homonuclear couplings (e.g., the coupling between pairs of 13C nuclei) are measured by matching the rotational frequency with the chemical shift difference of the interacting nuclei (Creuzet et al., 1991; Levitt et al., 1990; Spencer et al., 1994). This produces a transfer of magnetization between the interacting nuclei that can be measured with great precision, equivalent to distance measurement with subangstrom resolution. Measurement of 13C--13C distances is limited to 6 A or less. Recently, this technique was improved to gauge the longer intermolecular distances reliably (Balazs and Thompson, 1999). The REDOR experiments measures heteronuclear dipolar couplings, for example, the couplings between 15N and 13C nuclei in the peptide backbone. The method relies on the dephasing of magnetization of the observed spin through dipolar coupling to a second spin (Gullion and Schaefer, 1989). Accurate, highsensitivity measurement of distances in peptides has been achieved (Hing and Schaefer, 1993; Marshall et al., 1990). The method was used successfully to study the secondary structure of magainins (Hirsh etal., 1996). Measurement of 15N--13C distances is limited to 5 .~ or less. A complication of the original experiment is its sensitivity to the natural background signals of nuclei that are not participating in

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the dipolar interaction. Modifications allow the selection of dipole-coupled spins against a background of uncoupled spins (Holl et al., 1990). 3. Correlation of Anisotropy of Chemical Shift and Dipolar Interactions The purpose of the polarization inversion with spin exchange at the magic angle (PISEMA) experiment is to improve spectral resolution by correlating two orientation-dependent interactions, the 15N-chemical shift frequency and the strength of the IH--~SN dipolar coupling. PISEMA requires preparation of welloriented samples, which are investigated without magic angle spinning. The experiment is capable of resolving a large number of backbone 15N resonances, in particular for c~ helices that are oriented parallel to the magnetic field (Wu et al., 1994). To improve resolution, the experiment was extended to a third dimension by coupling 15N chemical shift, IH--15N dipolar interaction, and IH or X3Cchemical shift (Gu and Opella, 1999; Ramamoorthy et al., 1995). Transmembrane helices that are oriented parallel to the magnetic field form regular patterns in PISEMA spectra called polarity index slant angle (PISA) wheels (Wang et al., 2000). Location of resonances in the two-dimensional contour plots allows precise determination of helical tilt in the membrane (Marassi and Opella, 2000). After assignment of resonance signals, even the angle of rotational orientation of the helix can be determined (Wang et al., 2000). The method has great potential for the study of helix orientation in bundles of helices. 4. Measurement of ~b and ~b Torsion Angles of the Peptide Backbone Peptide secondary structure is linked to combinations of the stericatly permitted peptide backbone torsion angles ~b and 7r (Ramachandran et al., 1963) (see Fig. 4). The torsion angles can be measured by solid-state NMR experiments that correlate orientation-dependent interactions, such as the CSA of amide 15N, carbonyl 13C, and a-carbon 13C, or the dipolar coupling between pairs of nuclei, for exmple, lH--15N or 1H--13C~ (Feng et al., 1997; Reif et al., 2000; Weliky and Tycko, 1996). The 13C~-CSA for a-helical residues is smaller than for/~-sheet structures. This feature was used in a two-dimensional ~SN--13Ccorrelation experiment which selects the signals of helical residues (Hong, 2000). Correlation of relative orientations between IH--~SN and 1H~--13Cc~ bonds allows identification of/~-sheet residues (Huster et al., 2000). The torsion angle ap in :3C~ and carbonyl 13C doubly labeled amino acids was measured in experiments that correlate orientation of lH,~-13C~ bonds with the orientation of the carbonyl 13C-CSA tensor (Ishii et al., 1996; Schmidt-Rohr, 1996). Only one 13C-labeled amino acid per ~ angle is needed for this experiment. In contrast, distance measurements for determining peptide backbone structure by RR and REDOR experiments require labeling of two sequential residues. The experiment with

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double-quantum selection (Schmidt-Rohr, 1996) also suppresses 13C background signals and improves resolution by partially removing the inhomogenous spectral broadening of resonance lines.

11I. STRUCTURE A N D D Y N A M I C S OF THE LIPID MATRIX Peptide binding to membranes depends on the properties of the lipid matrix, and peptide binding alters lipid bilayer structure. In particular, the binding of amphipathic ct helices to the lipid/water interface has a profound influence on lipid organization. The lipid matrix of biomembranes belongs to the class of liquidcrystalline phases, which are very well suited for NMR structural studies. Lipid molecules have almost liquid-like degrees of freedom: vibrational changes of bond length and bond orientation with correlation times of a few picoseconds, gauche/trans isomerization of carbon-carbon bonds with correlation times on the order of 100 ps, molecular rotation and wobble with correlation times on the order of nanoseconds, and lateral diffusion of molecules within the plane of membranes at rates of D -~ 1 x 10- 8 cm 2 s-1 (Pastor and Feller, 1996) (see Fig. 5). However, all of these motions have a certain degree of spatial anisotropy. They reduce the magnitude of anisotropic interactions, but do not average them out entirely, as observed for molecules that perform isotropic tumbling motions. Therefore, dipole-dipole interactions, quadrupole interactions, and the anisotropies of

rotation xrt ~ 10-9 s

bond fluctuations "cB ~. 10"12 s

diffusion ~D ~ 10-7 S

trails gauche isomerization ~ts ~ 10"1° s

FIGURE 5 A biological membrane with phospholipid molecules and an attached amphiphatic peptide. Lipids in the membrane matrix exist in a multitude of conformations with rapid transition among them. They rotate, tumble, and perform rapid lateral diffusion. Correlation times that are of relevance for NOESY NMR are given. Motions of protein segments and of entire proteins are more restricted and much slower.

6. Lipid-Peptide Interaction Investigated by NMR

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chemical shift still broaden, split, and shift NMR resonance signals of lipids, but with reduced magnitude. The orientation of partially averaged interaction tensors is determined by the orientation of the symmetry axis of motions, for example, the normal to the lipid bilayer. The state of the lipid matrix is judged by the degree of averaging of anisotropic interactions.

A, 3l p Anisotropy o f Chemical Shift The 31p-anisotropy of the chemical shift (31P-CSA) of lipid phosphate groups provides information on the lipid phase state and on peptide interaction with the lipid/water interface. Typical rigid-lattice 31p-CSA values of phosphatidylcholines are 0-1l -81 ppm, a22 = - 2 5 ppm, and 0"33 108 ppm (convention: chemical shift values increase with increasing field strength) (Kohler and Klein, 1976). Values for phosphatidylethanolamines and phosphatidylserines are similar. Fast rotational diffusion of lipids about the bilayer normal and headgroup wobble reduce these values to a tensor with apparent axial symmetry, A0" = 0-11- 0-.1_~, - 4 5 ppm, w h e r e 0-11 is the tensor component oriented parallel to the bilayer normal and 0-± is the tensor component perpendicular to it (J. Seelig, 1978). Lipid motions in lamellar, inverse hexagonal, cubic, and micellar phases have different symmetry, resulting in easily distinguishable degrees of averaging of 31p-CSA. Therefore 31p NMR is a convenient tool for studying the phase state of lipids (Cullis and de Kruijff, 1978). Furthermore, 31p spectra enable the determination of mosaic spread in bilayer orientation for oriented membrane samples (Arnold et al., 1979). Interaction of peptides with the lipid/water interface alters the motionally averaged 31p-CSA, because of changes in headgroup orientation and changes in lipid phase state, and also by inducing membrane curvature in the lamellar phase. The fast lateral diffusion of lipids over lamellar surfaces with small radius of curvature is equivalent to lipid reorientation in the outer magnetic field. Effective anisotropies show first signs of a reduction for radii of curvature that are smaller than 5000 A. Anisotropies disappear completely for radii smaller than 1000 A, that is, for small unilamellar liposomes (Burnell et al., 1980; Gawrisch et al., 1986). The 13C atoms of lipid carbonyl groups have characteristic anisotropies of chemical shift as well. Effective values of carbonyl anisotropies of chemical shift are linked to the conformation and the mobility of the glycerol backbone in lipids (S. O. Smith et al., 1992). =

=

B. 2H N M R The prevalent approach to studying the conformation and dynamics of lipid molecules in membranes is specific deuteration of lipids combined with the

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measurement of 2H NMR quadrupole splittings. Very well resolved quadrupole splittings of deuterated lipid headgroups (Gally et al., 1975), hydrocarbon chains (A. Seelig and Seelig, 1975), and the glycerol backbone (J. Seelig, 1977) are detected. "High- fidelity" NMR spectra without baseline distortions and intensity and phase errors are obtained by the quadrupolar echo sequence (Davis et al., 1976). Experiments are mostly conducted on nonoriented lipid samples, but resolution of superimposed splittings and signal intensity improve considerably with the use of oriented membranes. The 2H NMR spectra of randomly oriented bilayers can be mathematically transformed into virtual spectra of oriented bilayers by a procedure called dePakeing (Sternin et al., 1983). More recently dePakeing was improved to analyze quadrupolar splittings and the distribution function of bilayer normals for samples that orient spontaneously in strong magnetic fields (Sch~ifer et al., 1998) and for oriented bilayers at the surface of solid substrates and in bicelles (Sternin et al., 2001). The value of the quadrupole splitting Av is conveniently expressed as an order parameter SCD that is linked to the orientation and motions of a C--D bond, Av = 3 ( e 2 Q / h ) S c D a n d S c D = (½(3 c o s 2 0 - 1)). The term e2Q/h is the quadrupole coupling constant, equal to 168 kHz for a typical C--D bond (Burnett and Muller, 1971), and 0 is the angle between the orientation of the C--D bond and the bilayer normal. The bracket indicates time averaging. The order parameter is +1 for bonds that are oriented parallel to the external magnetic field, vanishes to zero at a bond orientation of 54.7 ° (the magic angle), and is -0.5 for bonds oriented perpendicular to the magnetic field. Note that a reduced order parameter may indicate a particular orientation, but also motional averaging. For example, the decrease in chain order of lipid hydrocarbon chains toward the terminal methyl group has been linked to a probability of gauche-trans isomerization in chains that is higher at the terminal methyl end. Order in the upper half of saturated chains, that is, near the lipid/water interface, is high and almost constant (order parameter plateau), whereas the order in the second half of the chain decreases with a steep gradient toward the terminal methyl group (A. Seelig and Seelig, 1975). Chain order is very sensitive to changes in area per molecule, for example, as a result of changes in temperature or hydration (Koenig et al., 1997), or as the result of interaction with amphipathic peptides (Koenig et al., 1999a). Changes in lipid headgroup orientation have been followed via measurement of quadrupole splittings of choline a- and/%methylene groups (Gaily et al., 1975).

C. Magic Angle Spinning NMR A significant increase in the number of resolved lipid NMR resonances has been achieved by MAS. MAS NMR reduces the linewidth of lipid resonances to about

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10 Hz (Holte and Gawrisch, 1997), which is close to high-resolution NMR conditions. Resolution of lipid resonances in MAS experiments is better than resolution of spectra from very small unlilamellar liposomes, which tumble rapidly enough to eliminate anisotropic interactions. Magic angle spinning frequencies up to 10 kHz have negligible influence on lipid packing in bilayers. Experiments can be conducted at any level of hydration, provided that the lipids are in the biologically relevant liquid crystalline phase.

1. Dipolar Recoupling On-Axis with Scaling and Shape Preservation (DROSS) The two-dimensional DROSS experiment determines IH--13C dipolar interactions in lipid molecules (Gross et aL, 1997; Holte et al., 1998). The strength of dipolar interaction is a measure of the H--C bond order parameter, equivalent to order parameters determined by 2H NMR. Unlike 2H NMR, the DROSS experiment enables assignment of order parameters to lipid segments without specific labeling. The experiment takes advantage of the much greater chemical shift dispersion of 13C compared to 2H nuclei. Recoupling of dipolar interactions is achieved by application of radiofrequency pulses that are synchronized to the phase of the spinning rotor. For technical reasons, the precision of order parameters determined by DROSS is somewhat lower than the precision of order parameters determined by 2H NMR. 2. Nuclear Overhauser Enhancement Spectroscopy (NOESY) The excellent resolution of lipid IH NMR resonances (see Fig. 6) permits the application of techniques that probe magnetization transfer between protons. One such technique that is well known for its application to high-resolution NMR structural studies of soluble proteins is nuclear Overhauser enhancement spectroscopy (NOESY) (see Fig. 7). We developed MAS NOESY into a tool for membrane structural studies (Feller et al., 1999; Huster and Gawrisch, 1999; Huster et al., 1999; Yau and Gawrisch, 2000). Magnetization transfer is the result of interactions between the magnetic dipoles of the protons in lipids. Rates of transfer become observable when the protons approach each other to distances of 5 A or less. The surprising observation has been that magnetization is transferred between all lipid resonances, but at different rates. Even the most distant protons, such as methyl groups of the choline headgroup and methyl groups at the end of lipid hydrocarbon chains, exchange magnetization. Previously, this observation was explained by a process called spin diffusion, in which magnetization between protons is transferred indirectly as a multiple-step process. However, our experiments on protonated lipid in a deuterated matrix (Huster et al., 1999) and on binary mixtures of specifically deuterated lipids (Huster and Gawrisch, 1999) demonstrated unambiguously that spin diffusion is

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FIGURE 6 The IH MAS NMR spectrum of l-stearoyl-2-docosahexaenoyl-sn-glycero-3phosphocholine in 50 wt% D20 recorded at a resonance frequency of 500.13 MHz, a temperature of 25 °C, and an MAS spinning speed of 10 kHz. The resolution of 1HMAS spectra is better than the resolution of proton spectra from small unilamellar liposomes. insignificant under most experimental conditions. The direct intermolecular transfer of magnetization between lipid protons is more efficient. Cross-relaxation rates between lipid segments measured by IH N M R NOESY report the statistics" of contacts between neighboring lipid molecules. The only conceivable model that would allow the bewildering multitude of cross-relaxations to happen is one of a lipid bilayer with a very disordered arrangement of lipids (Holte and Gawrisch, 1997, 1999; Huster et al., 1999). Even protons that are separated by 20 lk or more, according to bilayer structures in the crystalline phase, can approach each other to less than 5/~ in the liquid crystalline phase, albeit with

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low probability. Precise quantitative interpretation of these headgroup-to-chain contacts is somewhat influenced by the secondary dependence of cross-relaxation on differences in correlation times and differences in proton-proton distances of closest approach. Measurement of 1H MAS NOESY cross-relaxation rates permits the study of lipid structure and dynamics in bilayers as well as lateral lipid organization in complex lipid mixtures (Huster et al., 1998). Furthermore, we successfully used this technique to locate small molecules, such as ethanol, and indole analogues in membranes with atomic resolution (Holte and Gawrisch, 1997; Yau et al., 1998).

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D. Influence o f Peptide Binding on Lipid Structure Interaction of peptides with the lipid matrix is reflected in changes of lipid order parameters. Amphipathic helical peptides have a preference for binding to the lipid/water interface region and tend to intercalate their hydrophobic side chains between lipid hydrocarbon chains to hide them from water. The partial penetration of the peptide into the lipid matrix increases area per lipid molecule at the lipid/water interface. The amino acid side chains are shorter than most lipid hydrocarbon chains and therefore do not reach to the bilayer center. This extra volume is filled by more-disordered hydrocarbon chain segments near the terminal methyl groups. It is reflected in a decrease of chain order parameters in 2H N M R experiments on perdeuterated hydrocarbon chains (see Fig. 8) (Koenig et al., 1999a). Depending on peptide penetration depth, such partial penetration creates positive membrane curvature stress, which has been linked to pore formation and membrane lysis.

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FIGURE 8 Influence of peptide P828S on the sn-1 hydrocarbon chain order of 1-stearoyl-d35-2oleoly-sn-glycero-3-phosphocholine(SOPC-d35) investigatedat 32°C. The 2H NMR order parameter profiles of SOPS-d35 in the absence of P828S (11) and at molar lipid : peptide ratios of 20 : 1 (O), and 10:1 (&), respectively, are shown in the upper panel. The peptide-induced difference in order parameters along the chain at molar lipid : peptide ratios of 20 : 1 (©), and 10 : 1 (A) is shown in the lower panel. Peptide-inducedorder changes are largest in the bilayer center, suggestingthat the peptide acts as a spacer that is located in the membrane's interface region. Figure reproduced from Koenig et al. (1999a).

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Charged amphipathic peptides have a tendency to change the orientation and flexibility of lipid headgroups, which is easily observed by 2H NMR on headgrouplabeled phospholipids and 31p NMR of lipid phosphate groups (Wieprecht et al., 2000). Lipid/water interface properties can be also conveniently studied via the 2H NMR quadrupole splittings of deuterated water (Gawrisch et al., 1992). Helical peptides that span the bilayer have less influence on the packing of lipid hydrocarbon chains. Membranes have a tendency to adjust their hydrophobic thickness to match the hydrophobic length of the peptide. Long peptides increase bilayer thickness and reduce area per lipid molecule, as reflected in an increase of chain order parameters. Peptides that are shorter have the opposite effect (de Planque et aI., 1998; Nezil and Bloom, 1992). Furthermore, peptides that are significantly shorter than the hydrophobic thickness of lamellar bilayers promote the formation of inverse-hexagonal and cubic lipid phases, which have been detected by 31p NMR measurements (Prenner et al., 1999; van der Wel et al., 2000).

IV. FUTURE DIRECTIONS The improvements in resolution and sensitivity of solid-state NMR experiments over the past 10 years are remarkable. However, it still takes considerable effort to synthesize the specifically labeled peptides required to obtain structural information from a sufficient number of peptide segments. High-resolution NMR on rapidly tumbling peptides provides a larger number of constraints in a single experiment. At the same time, the precision of distances, bond orientations, and dihedral bond angles measured by solid-state NMR is superior over equivalent values from high-resolution NMR measurements, enabling peptide structural determination by solid-state NMR with fewer constraints. Both techniques require special sample preparation procedures that may interfere with interpretation. High-resolution NMR relies on sufficiently fast isotropic motions of the peptide, whereas solid-state NMR requires peptide immobilization. The introduction of multidimensional experiments in combination with magic angle spinning, such as MAS NOESY, has increased resolution of lipid structural investigations. New insights into the conformational freedom of lipids, lateral lipid organization, and location of small membrane-bound molecules have been obtained. Future development of high-resolution approaches to peptide structural studies may benefit from utilizing bicelles that spontaneously orient in the strong magnetic fields applied to NMR samples (Sanders and Landis, 1995; Tjandra and Bax, 1997). High-resolution experiments on peptides that weakly interact with bicelles benefit from the small anisotropic NMR interactions, which can be measured without losing spectral resolution. Solid-state NMR experiments on peptides that are strongly bound to bicelles or incorporated into the bilayer region of bicelles

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benefit from the very high degree of bilayer orientation of bicelles in the magnetic field. Bicelles have the additional advantage that membrane surfaces are freely accessible from the water phase on both sides of the bilayer, enabling functional studies under almost physiological conditions. Acknowledgment K.G. thanks Dr. Daniel Huster for a valuable discussion on solid-state NMR approaches to studying peptide structure.

References Anglister, J., and Zilber, B. (1990). Antibodies against a peptide of cholera toxin differing in crossreactivity with the toxin differ in their specific interactions with the peptide as observed by 1H NMR spectroscopy. Biochemistry 29, 921-928. Arepalli, S. R., Glaudemans, C. E J., Daves, G. D., Kovac, E, and Bax, A. (1995). Identification of protein-mediated indirect NOE effects in a disaccharide-Fab ~ complex by transferred ROESY. J. Magn. Reson. B 106, 195-198. Amold, K., Gawrisch, K., and Volke, F. (1979). alp NMR investigations of phospholipids. I. Dipolar interactions and the 31p NMR lineshape of oriented phospholipid/water dispersions. Studia Biophys. 75, 189-197. Asakawa, N., Kuroki, S., Kurosu, H., Ando, I., Shoji, A., and Ozaki, T. (1992). Hydrogen-bonding effect on 13C NMR chemical-shifts of L-alanine residue carbonyl carbons of peptides in the solid-state. J. Am. Chem. Soc. 114, 3261-3265. Balazs, Y. S., and Thompson, L. K. (1999). Practical methods for solid-state NMR distance measurements on large biomolecules: Constant-time rotational resonance. J. Magn. Reson. 139, 371-376. Bax, A. (1989). Homonuclear Hartmann-Hahn experiments. Meth. Enzymol. 176, 151-168. Bax, A., Sparks, S. W., and Torchia, D. A. (1989). Detection of insensitive nuclei. Meth. Enzymol. 176, 134-150. Bechinger, B., and Opella, S. J. (1991). Flat-coil probe for NMR spectroscopy of oriented membrane samples. J. Magn. Reson. 95, 585-588. Bechinger, B., Kim, Y., Chirlian, L. E., Gesell, J., Neumann, J. M., Montal, M., Tomich, J., Zasloff, M., and Opella, S. J. (1991). Orientations of amphipathic helical peptides in membrane bilayers determined by solid-state NMR spectroscopy. J. Biomol. NMR 1, 167-173. Bechinger, B., Gierasch, L. M., Montal, M., Zasloff, M., and Opella, S. J. (1996). Orientations of helical peptides in membrane bilayers by solid state NMR spectroscopy. Solid State Nucl. Magn. Reson. 7, 185-191. Bersch, B., Koehl, P., Nakatani, Y., Ourisson, G., and Milon, A. (1993). 1H nuclear magnetic resonance determination of the membrane-bound conformation of senktide, a highly selective neurokinin-B agonist. J. Biomol. NMR 3, 443-461. Bogusky, M. J., Schiksnis, R. A., Leo, G. C., and Opella, S. J. (1987). Protein backbone dynamics by solid-state and solution 15N NMR spectroscopy. J. Magn. Resort. 72, 186-190. Bogusky, M. J., Leo, G. C., and Opella, S. J. (1988). Comparison of the dynamics of the membranebound form of fd coat protein in micelles and in bilayers by solution and solid-state nitrogen-15 nuclear magnetic resonance spectroscopy. Proteins 4, 123-130. Bothner-By, A. A., Stephens, R. L., Lee, J., Warren, Ch. D., and Jeanloz, R. W. (1984). Structure determination of a tetrasaccharide: Transient nuclear Overhauser effects in the rotating frame. J. Am. Chem. Soc. 106, 811-813. Brender, J. R., Taylor, D. M., and Ramamoorthy, A. (2001 ). Orientation of amide-nitrogen-15 chemical shift tensors in peptides: A quantum chemical study. J. Am. Chem. Soc. 123, 914-922.

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Burnett, L. J., and Muller, B. H. (1971). Deuteron quadrupole coupling constants in three solid deuterated paraffin hydrocarbons: C2D6, C4Dl0, C6D14..]. Chem. Phys. 55, 5829-5831. Burnell, E. E., Cullis, P. R., and de Kruijff, B. (1980). Effects of tumbling and lateral diffusion on phosphatidylcholine model membranes 31p NMR lineshapes. Biochim. Biophys. Acta 603, 63-69. Clore, G. M., and Gronenborn, A. M. (1982). Theory and application of the transferred nuclear Overhauser effect to the study of the conformations of small ligands bound to proteins. J. Magn. Reson. 48, 402-417. Clore, G. M., and Gronenborn, A. M. (1983). Theory of the time dependent transferred nuclear Overhauser effect: Applications to structural analysis of ligand-protein complexes in solution. J. Magn. Reson. 53, 423-442. Clore, G. M., and Gronenborn, A. M. ( 1991 ). Applications of three- and four-dimensional heteronuclear NMR spectroscopy to protein structure determination. Prog. NMR Spectrosc. 23, 43-92. Creuzet, E, McDermott, A., Gebhard, R., van der Hoef, K., Spijker-Assink, M. B., Herzfeld, J., Lugtenburg, J., Levitt, M. H., and Griffin, R. G. (1991). Determination of membrane-protein structure by rotational resonance NMR. Bacteriorhodopsin. Science 251, 783-786. Cullis, P. R., and de Kruijff, B. (1978). The polymorphic phase behaviour of phosphatidylethanolamines of natural and synthetic origin. A 31p NMR study. Biochim. Biophys. Acta 513, 31-42. Datema, K. P., Pauls, K. P., and Bloom, M. (1986). Deuterium nuclear magnetic resonance investigation of the exchangeable sites on gramicidin A and gramicidin S in multilamellar vesicles of dipalmitoylphosphatidylcholine. Biochemistry 25, 3796-3803. Davis, J. H., Jeffrey, K. R., Bloom, M., Valic, M. I., and Higgs, T. P. (1976). Quadrupolar echo deuteron magnetic resonance spectroscopy in ordered hydrocarbon chains. Chem. Phys. Lett. 42, 390-394. Dempsey, C. E., Cryer, G. D., and Watts, A. (1987). The interaction of amino-deuteromethylated melittin with phospholipid membranes studied by deuterium NMR. FEBS Lett. 218, 173-177. de Planque, M. R. R., Greathouse, D. V., Koeppe, R. E., Sch~er, H., Marsh, D., and Killian, J. A. (1998). Influence of lipid/peptide hydrophobic mismatch on the thickness of diacylphosphatidylcholine bilayers: A 2H NMR and ESR study using designed transmembrane alpha-helical peptides and gramicidin A. Biochemistry 37, 9333-9345. Deslauriers, R., and Smith, I. C. P. (1980). The multinuclear NMR approach to peptides. Structures, conformations, and dynamics. In "Biological Magnetic Resonance" (L. J. Berliner and J. Reuben, eds.), Vol. 2, pp. 243-344. Plenum Press, New York. Dyson, H. J., and Wright, P. E. (1991). Defining solution conformations of small linear peptides. Annu. Rev. Biophys. Biophys. Chem. 20, 519-538. Feller, S. E., Huster, D., and Gawrisch, K. (1999). Interpretation of NOESY cross-relaxation rates from molecular dynamics simulation of a lipid bilayer. J. Am. Chem. Soc. 121, 8963-8964. Feng, X., Eden, M., Brinkmann, A., Luthman, H., Eriksson, L., Graslund, A., Antzutkin, O. N., and Levitt, M. H. (1997). Direct determination of a peptide torsional angle psi by double-quantum solid-state NMR. J. Am. Chem. Soc. 119, 12006-12007. Forman-Kay, J. D., Gronenborn, A. M., Kay, L. E., Wingfield, P. T., and Clore, G. M. (1990). Studies on the solution conformation of human thioredoxin using heteronuclear 15N-IH nuclear magnetic resonance spectroscopy. Biochemistry 29, 1566-1572. Gaily, H. U., Niederberger, W., and Seelig, J. (1975). Conformation and motion of the choline head group in bilayers of dipalmitoyl-3-sn-phosphatidylcholine. Biochemistry 14, 3647-3652. Gawrisch, K., Stibenz, D., M6ps, A., Arnold, K., Linss, W., and Halbhuber, K. J. (1986). The rate of lateral diffusion of phospholipids in erythrocyte microvesicles. Biochim. Biophys. Acta 856, 443-447. Gawrisch, K., Ruston, D., Zimmerberg, J., Parsegian, V. A., Rand, R. P., and Fuller, N. (1992). Membrane dipole potentials, hydration forces, and the ordering of water at membrane surfaces. Biophys. J. 61, 1213-1223.

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Gawrisch, K., Han, K.-H., Yang, J.-S., Bergelson, L. D., and Ferretti, J. A. (1993). Interaction of peptide fragment 828-848 of the envelope glycoprotein of human immunodeficiency virus type I with lipid bilayers. Biochemistry 32, 3112-3118. Gesell, J., Zasloff, M., and Opella, S. J. (1997). Two-dimensional IH NMR experiments show that the 23-residue magainin antibiotic peptide is an alpha-helix in dodecylphosphocholine micelles, sodium dodecylsulfate micelles, and trifluoroethanol/water solution. J. Biomol. NMR 9, 127-135. Gross, J. D., Warschawski, D. E., and Griffin, R. G. (1997). Dipolar recoupling in MAS NMR: A probe for segmental order in lipid bilayers. J. Am. Chem. Soc. 119, 796-802. Gu, Z., and Opella, S. J. (1999). Three-dimensional 13C shift/IH-15N coupling/15N shift solid-state NMR correlation spectroscopy. J. Magn. Reson. 138, 193-198. Gullion, T., and Schaefer, J. (1989). Rotational-echo double-resonance NMR. J. Magn. Reson. 81, 196-200. Harbison, G. S., Jelinski, L. W., Stark, R. E., Torchia, D. A., Herzfeld, J., and Griffin, R. G. (1984). 15N chemical-shift and 15N_13C dipolar tensors for the peptide-bond in [ 1-13C]glycyl[ 15N]glycine hydrochloride monohydrate. J. Magn. Reson. 60, 79-82. Hing, A. W., and Schaefer, J. (1993). 2-Dimensional rotational-echo double-resonance of val-l [1-13C]gly2-[15N]ala3-gramicidin-A in multilamellar dimyristoylphosphatidylcholine dispersions. Biochemistry 32, 7593-7604. Hing, A. W., Adams, S. P., Silbert, D. E, and Norberg, R. E. (1990). Deuterium NMR of Vall... (23H)Ala3... gramicidin A in oriented DMPC bilayers. Biochemistry 29, 4144-4156. Hirsh, D. J., Hammer, J., Maloy, W. L., Blazyk, J., and Schaefer, J. (1996). Secondary structure and location of a magainin analogue in synthetic phospholipid bilayers. Biochemistry 35, 1273312741. Holl, S. M., McKay, R. A., Gullion, T., and Schaefer, J. (1990). Rotational-echo triple-resonance NMR. J. Magn. Reson. 89, 620-626. Holte, L. L., and Gawrisch, K. (1997). Determining ethanol distribution in phospholipid multilayers with MAS-NOESY spectra. Biochemistry 36, 4669-4674. Holte, L. L., Koenig, B. W., Strey, H. H., and Gawrisch, K. (1998). Structure and dynamics of the docosahexaenoic acid chain in bilayers studied by NMR and X-ray diffraction. Biophys. J. 74, A371. Hong, M. (2000). Solid-state NMR determination of 13C alpha chemical shift anisotropies for the identification of protein secondary structure. J. Am. Chem. Soc. 122, 3762-3770. Howard, K. E, and Opella, S. J. (1996). High-resolution solid-state NMR spectra of integral membrane proteins reconstituted into magnetically oriented phospholipid bilayers. J. Magn. Reson. B 112, 91-94. Huster, D., and Gawrisch, K. (1999). NOESY NMR crosspeaks between lipid headgroups and hydrocarbon chains: Spin diffusion or molecular disorder? J. Am. Chem. Soc. 121, 1992-1993. Huster, D., Arnold, K., and Gawrisch, K. (1998). Influence of docosahexaenoic acid and cholesterol on lateral lipid organization in phospholipid mixtures. Biochemistry 37, 17299-17308. Huster, D., Arnold, K., and Gawrisch, K. (1999). Investigation of lipid organization in biological membranes by two-dimensional nuclear Overhauser enhancement spectroscopy. Z Phys. Chem. B 103, 243-251. Huster, D., Yamaguchi, S., and Hong, M. (2000). Efficient beta-sheet identification in proteins by solid-state NMR spectroscopy. J. Am. Chem. Soc. 122, 11320-11327. Ishii, Y., Terao, T., and Kainosho, M. (1996). Relayed anisotropy correlation NMR: Determination of dihedral angles in solids. Chem. Phys. Lett. 256, 133-140. Ishima, R., and Torchia, D. A. (2000). Protein dynamics from NMR. Nat. Struct. Biol. 7, 740-743. Kay, L. E., and Bax, A. (1990). New methods for the measurement of NH--C,~H J couplings in 15N labeled proteins. J. Magn. Reson. 86, 110-126.

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