J. Phys. Chem. B 1999, 103, 8383-8390
8383
Application of One-Dimensional Dipolar Shift Solid-State NMR Spectroscopy To Study the Backbone Conformation of Membrane-Associated Peptides in Phospholipid Bilayers D. K. Lee,†,‡ J. S. Santos,† and A. Ramamoorthy*,†,‡ Biophysics Research DiVision and Department of Chemistry, The UniVersity of Michigan, Ann Arbor, Michigan 48109-1055 ReceiVed: May 5, 1999; In Final Form: August 3, 1999
A simple one-dimensional dipolar shift solid-state NMR experiment is demonstrated to study the backbone conformation of membrane-associated peptides embedded in phospholipid bilayers. The nitrogen-15 chemical shift and 1H-15N dipolar coupling parameters are measured on a magainin peptide selectively labeled with a 15N isotope at the Gly-18 site. Fully hydrated multilamellar vesicles and uniaxially oriented bilayer samples are used to determine the orientation of the peptide plane relative to the direction of the external magnetic field. It is inferred that the 1H-15N dipolar coupling 15N chemical shift doublet of the [15N-Gly-18]magainin peptide oriented in lipid bilayers is asymmetrical. Calculation of the experimental dipolar shift spectrum suggests that the shape of the asymmetrical doublet is highly sensitive to the orientation of the principal axes of the 15N chemical shift tensors in the molecular frame as well as the backbone conformation of the peptide embedded in lipid bilayers.
Introduction Membrane-associated proteins, integral parts of cell membranes, are responsible for a variety of biological functions, receiving and transducting signals from the environment, transport for appropriate molecules into or out of the cell, catalysis of chemical reactions, etc. Structure determination of membrane proteins is one of the important challenges in the field of structural biology at the present time. While X-ray crystallography and solution NMR spectroscopy have been successful at determining globular protein structures, they have faced numerous difficulties in producing equal levels of insight into the structure and dynamics of membrane-associated proteins. Solid-state NMR spectroscopy, although it is still in statu nascendi, is emerging as a method capable of describing the structures and dynamics of proteins.1-7 In one of the approaches,2 orientational constraints for bonds and chemical groups are derived from spectral parameters observed in proteins embedded in uniaxially oriented lipid bilayers. Recently, several solid-state NMR multidimensional methods have been successfully used to measure the 15N chemical shifts and 1H-15N dipolar couplings from polypeptides that are selectively as well as nonselectively labeled with 15N isotopes.3,5,7 However, these methods are time-consuming, and therefore one should be cautious in applying them to samples that are sensitive to heating due to radio-frequency (rf) power. In this paper, we demonstrate the applications of a simple one-dimensional method8 to study the backbone conformation of a membrane-associated peptide that is selectively labeled with 15N isotope at a single site. In the recent past, short synthetic peptides (20-30 residues) embedded in phospholipid bilayers have been used as model systems for the development of solid-state NMR spectroscopy to investigate the structural biology of membrane-associated proteins.2,7 The samples used in the present work consisted of †
Biophysics Research Division. Department of Chemistry. * To whom correspondence should be addressed. Phone (734)647-6572; FAX (734)764-8776; E-mail
[email protected]. ‡
a specifically 15N-labeled magainin2 peptide. The sequence of magainin2 is n-GIGKFLHSAKKFGKAFVGEIMNS-amide.9 The magainins are a family of 21-26 residue amphipathic helical peptides, found originally in frog skins, which have potent antibiotic activities.9 There is a strong evidence that their biological activities are due to interactions with membranes rather than protein receptors.9 Structural studies using solidstate and solution NMR methods predict that magainin2 is helical in several membrane environments.7,10-12 Experimental Section Fmoc-glycine amino acid labeled with 15N was purchased from Cambridge Isotope Laboratory (Cambridge, MA). All other amino acids were purchased from PerSeptive Biosystems (Framingham, MA). Phospholipids were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL). Both amino acids and lipids were used without further purification. Magainin2 peptide was synthesized with 15N-glycine at position 18 using fluorenylmethoxycarbonyl (Fmoc) blocking chemistry and an automated solid-phase peptide synthesizer. Four different types of samples (represented as samples I-IV) were used in the experimental studies in this work: sample I, a powder sample of the pure synthetic [15N-Gly-18]-magainin2 peptide; sample II, a dry powder sample containing phospholipids (80% palmitoyloleoylphosphatidylcholine (POPC) and 20% palmitoyloleoylphosphatidylglycerol (POPG)) and the synthetic [15N-Gly-18]magainin2 peptide; sample III, fully hydrated multilamellar vesicles containing phospholipids (80% POPC and 20% POPG) and the synthetic [15N-Gly-18]-magainin2 peptide; sample IV, a uniaxially oriented bilayer sample containing phospholipids (80% POPC and 20% POPG) and the synthetic [15N-Gly-18]magainin2 peptide. Seven milligrams of the peptide was cosolubilized in chloroform and trifluoroethanol along with POPC (80%) and POPG (20%). The peptide-lipid mixture (with 3% molar ratio of peptide to lipids) was then spread on the surface of thin glass plates, thoroughly cleaned with chloroform. Twenty com-
10.1021/jp9914929 CCC: $18.00 © 1999 American Chemical Society Published on Web 09/11/1999
8384 J. Phys. Chem. B, Vol. 103, No. 39, 1999 mercially purchased glass plates each with a thickness of 0.05 mm and a dimension of 11 × 11 mm were used. After evaporating the solvents from the material deposited on the glass plates under a stream of nitrogen gas, more of the solution containing the peptide and lipids was spread on top of the dried material. This procedure was repeated until the total peptidelipid mixture was uniformly distributed on top of the glass plates. Residual traces of the organic solvents were removed thoroughly under high vacuum for about 24 h at room temperature. After that, the plates were stacked, and a clean glass plate was placed on top of the sandwich sample. The sample was then hydrated in a chamber equilibrated at 98% relative humidity (with a saturated (NH4)H2PO4 solution) at room temperature for 2 days. For NMR experiments the sample was wrapped in Parafilm before insertion into the rf coil of the probe. A four-turn square coil made of a flat wire, with a width of 2 mm and a spacing of 1 mm, was used in a home-built double-resonance (1H and 15N) solid-state NMR probe to perform experiments on an oriented sample. Experiments were performed with the bilayer normal of the oriented sample (sample IV) in the NMR probe parallel to the external magnetic field of the spectrometer. Unoriented samples (samples II and III) were prepared from a solution containing the peptide and phospholipids (80% POPC and 20% POPG) with 10% molar ratio of peptide to lipids. All of the experiments were performed on a Chemagnetics Infinity 400 MHz solid-state NMR spectrometer operating at a field of 9.4 T with resonance frequencies 400.14 and 40.54 MHz for 1H and 15N, respectively. All of the magic angle spinning (MAS) experiments were performed using a Chemagnetics doubleresonance MAS probe. Typical 90° pulse lengths were 3.0-3.7 and 3.5-4.8 µs for 1H and 15N, respectively. All spectra were obtained using the cross-polarization sequence with a contact time of 2 ms. After the cross-polarization, the 15N magnetization was refocused by a 180° pulse to overcome the difficulties due to the receiver dead time, and the second half of the spin-echo signal was acquired. For experiments under MAS conditions, a two-pulse phase-modulated (TPPM) decoupling sequence13 was used to decouple protons during the 15N signal acquisition. A recycle delay of 5 s was used. For the one-dimensional dipolar chemical shift experiment,8 the frequency of proton decoupling was shifted by an offset in order to establish an effective field at the magic angle.14,15 This magic angle rf irradiation of protons suppresses the 1H-1H homonuclear dipolar interactions, and thus the resultant one-dimensional spectrum consists of the 15N chemical shift and 1H-15N dipolar interactions. An experimentally determined scaling factor, 0.56 ( 0.02, for the magic angle rf irradiation was used in the calculations of the dipolar shift spectra. All the 15N spectra are referenced relative to NH3 (liquid, 25 °C) by setting the observed 15N signal of the saturated aqueous NH4Cl solution to 27.3 ppm.16 Simulations. Calculations of the one-dimensional chemical shift and dipolar shift powder patterns and the oriented magainin2 spectra were carried out using a FORTRAN-77 program in a Macintosh computer. The magnitudes of the principal values (σ11N, σ22N and σ33N) of the 15N chemical shift tensor were obtained from the frequencies of the discontinuities of a powder pattern, and these principal values were used as starting parameters to simulate the chemical shift spectrum of a powder sample. The resultant simulated powder patterns were varied on the basis of the direct comparison with the respective experimental spectra in order to obtain the best-fitting result. The principal elements of the chemical shift tensors are represented according to the convention σ33N g σ22N g σ11N.
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Figure 1. Orientations of the principal axes of the 15N chemical shift tensor relative to the direction of the external magnetic field, B0, and to the N-H bond. θ is the angle between σ33N and the magnetic field axis (z axis). φ is the angle between σ11N and the projection of B0 on the σ11N-σ22N plane. RN is the angle between σ11N and the projection of the N-H bond on the σ11N-σ22N plane. βN is the angle between σ33N and the N-H bond.
Coordinates of the chemical shift and dipolar coupling tensors are defined in Figure 1. The dipolar chemical shift spectrum contains contributions from both the 15N chemical shift as well as the scaled dipolar coupling frequencies, and they are given as
δcS ) σ11N cos2 φ sin2 θ + σ22N sin2 φ sin2 θ + σ33N cos2 θ (1) 0.58 ωIS ) DIS[1 - 3{sin θ sin βN cos(φ - RN) + 2 cos θ cos βN}2] (2) where δCS is the chemical shift, ωIS is the dipolar coupling between I and S nuclei, and DIS is given as µ0pγIγS/4πrIS3. The angles θ and φ are defined in Figure 1. Dipolar chemical shift powder patterns were simulated using the principal values of the 15N chemical shift tensor and interactively varying the angles RN and βN for a fixed value of N-H bond length. The whole procedure to find out the best-fitting spectrum was then repeated by varying the N-H bond length from 1.01 to 1.1 Å in steps of 0.01 Å. This range for the N-H bond length was selected on the basis of the NMR studies reported in the literature.8,17-20 All the spectral lines were assumed to be Gaussian. Calculation of the spectral features does not include any specific relaxation effects, but a Gaussian line broadening was used in the simulation of chemical shift and dipolar shift spectra, respectively. The best-fitting simulated spectra were obtained by comparing the ratios of the intensities of the shoulders and their frequency separations with the experimental powder pattern spectra. In the case of the oriented sample, the 15N chemical shift frequency of the oriented sample as well as the magnitudes of the 15N chemical shift tensor (σ11N, σ22N, and σ33N) determined from the powder sample was used to calculate θ and φ angles from the eq 1 as explained in the following section. More details on the simulation of solid-state NMR spectra from uniaxially aligned samples can be found in ref 21. Results and Discussion The experimental 15N chemical shift spectrum of a powder sample of [15N-Gly-18]-magainin2 peptide in its pure form
Backbone Conformation of Peptides
Figure 2. Nitrogen-15 chemical shift spectra of a powder sample of [15N-Gly-18]-magainin2 peptide (sample I) under the static (experimental (A) and simulated (B)) and spinning (C) conditions. The simulated spectrum (B) was obtained using the parameters σ11N ) 42.3 ( 2 ppm, σ22N ) 72.7 ( 3 ppm, and σ33N ) 215.3 ( 2 ppm. (D) and (E) are the experimental and simulated 1H-15N dipolar 15N shift spectra of [15N-Gly]-magainin2 peptide, respectively. The simulated spectrum (E) was obtained with σ11N ) 42.3 ( 2 ppm, σ22N ) 72.7 ( 3 ppm, σ33N ) 215.3 ( 2 ppm, 1H-15N dipolar coupling constant ) 11.2 ( 0.4 kHz, RN ) 30 ( 10°, and βN ) 22 ( 2°. Spectra A, C, and D are the resultant of 3000, 72, and 5000 scans, respectively, with a recycle delay of 5 s. Spectra A, C, and D were processed with a Gaussian line broadening of 200, 100, and 800 Hz, respectively. Experimental spectra obtained at -150 °C and at room temperature are the same.
(sample I) is shown in Figure 2A. The powder patterns obtained at room temperature and at -150 °C were identical. This result suggests that there is no molecular motion in the peptide that can be observed on the NMR time scale. Therefore, the 15N chemical shift and the 1H-15N dipolar coupling tensors can be determined at room temperature without any errors due to molecular motions. The principal values σ11N, σ22N, and σ33N of the 15N chemical shift tensor 42.3 ( 2, 72.7 ( 3, and 215.3 ( 2 ppm, respectively, were used to calculate the best-fitting 15N chemical shift spectrum in Figure 2B. An experiment under MAS with a spinning frequency of 4 kHz displays a line having 7 ppm full width at half-height (see Figure 2C). The unusual line broadening may be attributed to the presence of multiple crystal forms of the peptide. This could also be the reason for the broad shoulders of the chemical shift powder pattern in Figure 2A. However, the isotropic 15N chemical shift frequency (110.1 ppm) obtained from the MAS experiment, within experimental errors, is identical to the value determined from the average of the principal values of the chemical shift tensors. Parts D and E of Figure 2 respectively are experimental and calculated one-dimensional spectra consisting of 1H-15N dipolar coupling as well as 15N chemical shift (abbreviated as dipolar shift) of the powder sample of [15N-Gly-18]-magainin2 peptide. The best-fitting calculated spectrum in Figure 2E was obtained using eqs 1 and 2 with the following parameters: σ11N ) 42.3 ppm, σ22N ) 72.7 ppm, σ33N ) 215.3 ppm, RN ) 30 ( 10°, and βN ) 22 ( 2°. A 1H-15N dipolar coupling constant, D ) µ0γNγHh/4πrNH3, of 11.15 ( 0.4 kHz corresponding to the N-H bond length of 1.03 ( 0.01 Å was used in the simulation. This bond length is in good agreement with most of the NMR studies reported in the literature.17-20 On the other hand, rNH ) 1.03 Å is longer than the values measured from diffraction studies.22 Dipolar shift powder patterns generated as a function of one angle (RN or βN) while keeping the second angle fixed are given in Figures 3 and 4. It is evident from Figures 3 and 4 that the
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Figure 3. Simulated one-dimensional 1H-15N dipolar 15N shift spectra of a powder sample of [15N-Gly]magainin2 peptide (sample I) for various values of βN with RN ) 30°,150°,210°, or 330°. All other parameters are as mentioned in the Figure 2 caption. Powder patterns spectra for βN ) (x° values are identical.
Figure 4. Simulated one-dimensional 1H-15N dipolar 15N shift spectra of a powder sample of [15N-Gly]magainin2 peptide (sample I) for various values of RN with βN ) 22°. All other parameters are as mentioned in the Figure 2 caption. Powder pattern spectra for RN ) (x° and 180 ( x° values are identical.
dipolar shift powder pattern is highly sensitive to the changes in the βN value and comparatively less sensitive to the changes in the RN value. The best-fitting spectrum in Figure 2E was obtained by comparing the frequency separation of the shoulders as well as the ratio of the intensity of the shoulders between the experimental and simulated spectra. For example, although the appearance of the spectra in Figure 4 for RN values ranging from 0° to 40° resembles the experimental spectrum in Figure 2D, the frequency separation of the two shoulders at the lowfield region of the experimental spectrum matches only with the simulated spectrum for RN ) 30°. It is clear from Figure 4 that the frequency of the shoulder at 362 ppm does not depend on the value of the RN angle, but the shoulder indicated by a dashed vertical line changes from 152 ppm for RN ) 0° to 122 ppm for RN ) 90°. But a large error ((10°) in the estimation of the RN value is due to the unresolved hump near the shoulder at 155 ppm in the experimental spectrum. According to the definition (see Figure 1), the angle RN varies from 0° to 360° and the angle βN varies from 0° to 180°. For a given value of RN, dipolar shift powder patterns are identical for βN ) (x° values. On the other hand, for a given value of
8386 J. Phys. Chem. B, Vol. 103, No. 39, 1999
Figure 5. Nitrogen-15 chemical shift spectra of unoriented (A) and uniaxially oriented (B) bilayer samples containing [15N-Gly-18]magainin2 peptide and phospholipid bilayers (80% POPC and 20% POPG). Experimental spectra A and B are the resultant of 3600 scans at -150 °C and 15 000 scans at room temperature, respectively, with a recycle delay of 5 s. Experimental spectra A and B were processed with a Gaussian line broadening of 100 and 250 Hz, respectively. Calculated spectra are shown as dashed lines. Calculated spectrum in (B) was obtained using σ11N ) 40.3 ( 2 ppm, σ22N ) 58.8 ( 2 ppm, σ33N ) 220.8 ( 2 ppm, θ ) 69°, and φ ) 28°. A variation of 3° in θ and φ angles is given in the Gaussian function to account for the uncertainty in the distribution of the orientation of the peptide in lipid bilayers.
βN, dipolar shift powder patterns are identical for RN ) (x° and 180 ( x° values. Therefore, the angles defining the orientation of the 15N chemical shift tensor elements predicted from the powder pattern spectra are RN ) 30 ( 10°, 150 ( 10°, 210 ( 10°, or 330 ( 10° and βN ) 22 ( 2° or 158 ( 2°. On the basis of the results from a single-crystal study, we assumed that σ33N is in the peptide plane.23 Our results predict that σ33N is 22 ( 2° (or 158 ( 2°) away from the N-H bond, σ11N is 30 ( 10° (or 150 ( 10°, 210 ( 10°, or 330 ( 10°) away from the peptide plane, and σ22N is 30 ( 10° (or 150 ( 10°, 210 ( 10°, or 330 ( 10°) away from the normal to the glycine peptide plane. Most of the previous studies based on the analysis of the results from a single crystal23 and powder samples24-38 assumed that σ11N is in the peptide plane and σ22N is normal to the peptide plane; some of these studies27-29,35 reported a large error ((5-10°) in the RN angle, indicating that a nonzero RN value is also possible. In contrast, the orientations of the σ11N and σ22N axes predicted from the present work are in agreement with the recent studies reported in the literature.17,18,39 A nonzero RN value determined for the terminal residues (Val1 and Gly2) of the gramicidin peptide embedded in lipid bilayers35 and the results from the quantum calculations40 also support the results presented in this paper. Parts A and B of Figure 5 show the 15N chemical shift spectra of multilamellar vesicles (sample III) and uniaxially oriented bilayers (sample IV), respectively; experimental and simulated spectra are given as solid and dashed lines, respectively. The 15N chemical shift value, 67.8 ( 3.5 ppm, obtained from the oriented spectrum in Figure 5B is consistent with the values reported in the literature.11 The powder pattern in Figure 5A was simulated with σ11N ) 40.3 ( 2 ppm, σ22N ) 58.8 ( 2 ppm, and σ33N ) 220.8 ( 2 ppm. These principal values were used to calculate the 15N chemical shift spectrum of the oriented sample. The possible values of θ and φ angles calculated using the 15N chemical shift parameter are presented in Figure 6. Even though the angle φ varies from 0° to 360°, for a given value of
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Figure 6. Possible values of θ and φ angles calculated using the 15N chemical shift frequency of the oriented bilayer sample (sample IV), 67.8 ( 3.5 ppm (obtained from Figure 5B), and the magnitudes of the principal elements of the 15N chemical shift tensor, σ11N ) 40.3 ppm, σ22N ) 58.8 ppm, and σ33N ) 220.8 ppm (obtained from Figure 5A).
θ the value of φ is restricted to a narrow range as shown in Figure 6. For example, if θ is 69°, then the value of φ ranges from -30° to 30° and 150° to 210°. Our calculations suggest that the value of φ can be further restricted if the line shapes of the calculated and experimental 15N chemical shift spectra are directly compared instead of using the magnitude of the 15N chemical shift parameter alone. As has been demonstrated in the literature,5,7 multiple experimental parameters are essential to determine the angles θ and φ accurately. Figure 7 shows the dipolar shift spectrum of [15N-Gly-18]magainin2 obtained from multilamellar vesicles at -100 °C (Figure 7A,B) and from the uniaxially oriented bilayers at room temperature (Figure 7C,D). The best-fitting powder pattern in Figure 7B was obtained using σ11N ) 40.3 ( 2 ppm, σ22N ) 58.8 ( 2 ppm, σ33N ) 220.8 ( 2 ppm, dipolar coupling constant ) 9.97 ( 0.6 kHz that corresponds to rNH ) 1.07 ( 0.02 Å, RN ) 40 ( 15°, and βN ) 24 ( 3°. Since the signal-to-noise ratio of the experimental spectrum (see Figure 7A) is poor and the shoulder in the low field region (near 300 ppm) is not well resolved, the frequency separation and the intensity ratio of the shoulders near 120 and 0 ppm were used in obtaining the bestfitting simulated spectrum (see Figure 7B). This resulted in a large error for the calculated values of rNH ((0.02 Å) and the angles (RN and βN) as compared to the results obtained from Figure 2C,D. The dipolar shift spectrum of the uniaxially oriented bilayer sample (sample IV) presented in Figure 7C shows an asymmetrical doublet centered at the 15N chemical shift value, 67.8 ppm, of the oriented sample. The best-fitting simulated spectrum shown in Figure 7D was obtained using σ11N ) 40.3, σ22N ) 58.8, σ33N ) 220.8 ppm, the 1H-15N dipolar coupling constant ) 9.97 kHz, θ ) 69°, φ ) 28°, RN ) 140 ( 15°, and βN ) 24 ( 3°. The asymmetry in the doublet spectral lines (see Figure 7C,D) is because of the 3° variation in θ and φ angles that was used to account for the uncertainty in the distribution of the orientation of the peptide in lipid bilayers. Using the equation DNH ) D(3 cos2 ΘNH - 1), the angle between B0 and the N-H bond (ΘNH) is calculated to be 80 ( 2°, where DNH is the frequency separation of the peaks in the dipolar shift doublet. The values of the angles θ and φ were arbitrarily chosen to calculate the spectrum in Figure 7D. However, any values of θ and φ chosen from the restriction plot (see Figure 6) resulted in the best-fitting spectrum (see Figure 7D) with the same values of angles RN (140 ( 15°) and βN (24 ( 3°), except when φ is (10°, 90 ( 10°, or 180 ( 10°. When using these exceptional φ values, the value of RN has to be changed from 140° to 0° in order to obtain the best-fitting
Backbone Conformation of Peptides
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Figure 8. Calculated 1H-15N dipolar 15N shift spectra of [15N-Gly16]-magainin2 peptide oriented in phospholipid bilayers (sample IV) for various values of βN and RN ) 140°. All other parameters are as mentioned in the Figure 7 caption.
Figure 7. Experimental (A and C) and calculated (B and D) 1H-15N dipolar 15N shift spectra of [15N-Gly-18]-magainin2 peptide embedded in phospholipid bilayers. Spectra from multilamellar vesicles (sample III) are given in (A) and (B), and the spectra from an oriented sample (sample IV) are given in (C) and (D). Experimental spectra A and C are the resultant of 20 000 scans at -100 °C and 50 000 scans at room temperature, respectively, with a recycle delay of 5 s. Experimental spectra A and C were processed with a Gaussian line broadening of 800 and 700 Hz, respectively. Simulated spectrum B was obtained using σ11N ) 40.3 ppm, σ22N ) 58.8 ppm, σ33N ) 220.8 ppm, and 1H-15N dipolar coupling constant ) 9.97 ( 0.6 kHz, RN ) 40 ( 15°, and βN ) 24 ( 3° parameters. Simulated spectrum D was obtained using σ11N ) 40.3 ppm, σ22N ) 58.8 ppm, σ33N ) 220.8 ppm, θ ) 69°, φ ) 28°, 1H-15N dipolar coupling constant ) 9.97 ( 0.6 kHz, R ) 140 ( N 15°, and βN ) 24 ( 3° parameters. A variation of 3° in θ and φ angles is given in the Gaussian function to account for the uncertainty in the distribution of the orientation of the peptide in lipid bilayers.
spectrum in Figure 7D. Therefore, the tensors are highly dependent on the orientation of the peptide plane or the backbone conformation of the peptide. It is worth mentioning here that the asymmetry of scalar coupled doublets has been analyzed in detail and reported in the literature based on the relaxation study of small molecules,41-47 RNA,48,49 and proteins50-60 in solution. These studies have demonstrated that the cross-correlation between 1H-15N dipolar interaction and 15N chemical shift anisotropy (CSA) gives rise to different transverse relaxation rates of the doublet components of the dipolar shift spectrum.51 Relaxation interference effects between CSA and dipolar interaction contain information on motional properties and chemical shift tensors.51-60 In fact, quantitative measurement of cross-correlation effects between 15N CSA and 1H-15N dipolar interaction, 1H CSA and 1H15N dipolar interaction, and 13C CSA and 1H-13C dipolar R R interaction is currently being used to characterize the backbone dynamics of proteins in solution.51-60 Recently, Fushman and Cowburn60 have analyzed the effect of noncollinearity of 1H15N dipolar and 15N CSA tensors and rotational anisotropy on 15N relaxation, CSA/dipolar cross-correlation, and TROSY.57 Our simulations predict that the shape of the dipolar shift spectral lines depends on the 15N chemical shift and 1H-15N dipolar coupling tensors and the distribution of the orientation of the peptide in lipid bilayers. In the calculation of the asymmetrical doublet as well as in the dipolar shift powder pattern spectra, we have not considered any specific relaxation effects. If the
Figure 9. Calculated 1H-15N dipolar 15N shift spectra of [15N-Gly16]-magainin2 peptide oriented in phospholipid bilayers (sample IV) for various values of RN and βN ) 24°. All other parameters are as mentioned in the Figure 7 caption.
cross-relaxation is effective, then it should play the same role in the dipolar shift spectra obtained from both the powder and uniaxially oriented samples. The fact that the results from the powder and oriented samples are the same, within the experimental error, suggests that the cross-relaxation effects within the resolution and time scale of solid-state NMR may be negligible. Therefore, the spin interaction tensors reported in this work will be highly valuable for cross-correlation studies in solution NMR spectroscopy. The dipolar shift spectra of uniaxially oriented bilayers (sample IV) are highly sensitive to the variation of RN and βN angles as shown in Figures 8 and 9. The values of RN and βN angles determined independently from samples I, II, and III are in good agreement; the value RN ) 270° was neglected, even though it fits well with the experimental dipolar shift spectrum of the oriented bilayer sample (see Figure 9), as it does not
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TABLE 1: 15N Chemical Shift and 1H-15N Dipolar Coupling Tensors of Magainin2 Peptide Labeled with 15N Isotope at the Glycine-18 Site sample
σ11N (ppm)
σ22N (ppm)
σ33N (ppm)
σiso (ppm)
RN (deg)
βN (deg)
DNH (kHz)a
rNH (Å)
I II III IV
42.3 ( 2 45.2 ( 2 40.3 ( 2 40.3 ( 2
72.7 ( 3 63.9 ( 3 58.8 ( 2 58.8 ( 2
215.3 ( 2 218.7 ( 2 220.8 ( 2 220.8 ( 2
110.1 109.3 106.6 106.6
30 ( 10 35 ( 10 40 ( 15 140 ( 15
22 ( 2 24 ( 2 24 ( 3 24 ( 3
11.2 ( 0.4 10.5 ( 0.4 9.9 ( 0.6 9.9 ( 0.6
1.03 ( 0.01 1.05 ( 0.01 1.07 ( 0.02 1.07 ( 0.02
a
Dipole coupling constant, DNH, (µ0γHγNp/4πrNH3).
Figure 10. Calculated 1H-15N dipolar 15N shift spectra of [15N-Gly16]-magainin2 peptide oriented in phospholipid bilayers (sample IV) for various values of θ. All other parameters are as mentioned in the Figure 7 caption.
Figure 11. Calculated 1H-15N dipolar 15N shift spectra of [15N-Gly16]-magainin2 peptide oriented in phospholipid bilayers (sample IV) for various values of φ. All other parameters are as mentioned in the Figure 7 caption.
TABLE 2: Variation of rΝ and βΝ Angles Resulting from the Symmetry-Related Values of θ and O
spectrum yielded RN ) 40 ( 15°. Therefore, consideration of indistinguishable values for the φ angle, that is φ ) (x° and 180 ( x°, results in RN values that are symmetric with respect to 0° and 180° as presented in Table 2. Therefore, the unique value of RN can be determined only if the conformation of the peptide plane or the backbone conformation of the peptide is uniquely determined. The dipolar shift spectra of the oriented bilayer sample calculated for various values of θ and φ angles are shown in Figures 10 and 11 for the angles RN ) 140° and βN ) 24°. The values 0-67°, 77-103°, and 113-180° for the angle θ are not taken into account as they do not fit the 15N chemical shift spectrum of the oriented bilayer sample (sample IV). It is clear from Figure 10 that the dipolar shift spectra for θ ) (x° values are entirely different. Similarly, the results shown in Figure 11 suggest that the dipolar shift spectra for φ ) (x° and φ ) 180 ( x° values are entirely different. The value φ ) 240° was neglected, even though it fits well with the experimental dipolar shift spectrum (see Figure 11), as it does not fit the 15N chemical shift spectrum of the oriented bilayer sample (see Figure 6). The principal values of the 15N chemical shift tensor obtained for the [15N-Gly-18]-magainin2 peptide in four different forms (samples I-IV) are given in Table 1. It is interesting to note that the magnitude of the principal elements of the 15N chemical shift tensor calculated from the pure powder sample of the peptide (sample I) is significantly different from the one obtained from the fully hydrated multilamellar vesicles (sample III).
θ (deg)
φ (deg)
RΝ ((15°)
βΝ ((3°)
69 69 69 69 111 111 111 111 69 69 69 69 111 111 111 111
28 152 208 332 332 208 152 28 28 152 208 332 332 208 152 28
140 40 320 220 40 140 220 320 140 40 320 220 40 140 220 320
24 24 24 24 24 24 24 24 156 156 156 156 156 156 156 156
agree with the results predicted from the dipolar shift powder patterns. However, unlike the unoriented powder patterns, spectra for the oriented sample (sample IV), with RN ) (x° and 180 ( x° values for a fixed value of βN and with βN ) (x° values for a fixed value of RN, are entirely different when the values of θ and φ are fixed. For example, when θ ) 69° and φ ) 28°, the calculation of the oriented spectrum yielded RN ) 140 ( 15°, thus ruling out -140°, 40°, and 320° values for the RN angle. But when φ ) 152°, the calculation of the oriented
Backbone Conformation of Peptides Changes in σ11N, σ22N, and σ33N are 2, 13.9, and 5.5 ppm, respectively. In particular, σ22N is highly sensitive to the environment of the peptide in the sample. It is worth mentioning here that CPMAS studies on copolypeptides have proven that the value of σ22N is governed mainly by secondary structures rather than by the amino acid sequences of polypeptides.61 Therefore, the sensitivity of σ22N found in the [15N-Gly-18]magainin2 peptide may be attributed to the conformation changes in the system. For example, according to the studies reported in ref 61, the value σ22N ) 58.8 ppm predicts that the magainin2 peptide is R-helical in phospholipid bilayers. On the other hand, the values obtained from the unhydrated powder samples (samples I and II) do not predict an R-helical conformation for the peptide. These results are in complete agreement with the reported secondary structure of the magainin2 peptide in membrane environments.11 Further, our calculations predict that the 15N chemical shift as well as the 1H-15N dipolar coupling frequencies of an oriented sample (sample IV) has a linear dependency on σ22N. This indicates that any error in the measurement of σ22N value will significantly change the predicted conformation of the peptide plane in lipid bilayers. Therefore, it is important to characterize the chemical shift tensors in the system of interest rather than relying on the data from model compounds for the structural studies of polypeptides. Conclusions It was demonstrated that a simple one-dimensional dipolar shift experiment is useful to characterize spin interaction tensors in the molecular frame and to study the backbone conformation of peptides oriented in phospholipid bilayers. Our results suggest that the spin interaction tensors are dependent on the molecular system as well as on the secondary structure of the molecule. Therefore, tensors of relevant sites have to be obtained in native or near native environments. These conclusions have led us to suggest that the one-dimensional dipolar shift spectroscopy will greatly improve the reliability of peptide 15N chemical shift anisotropy and 1H-15N dipolar coupling in the determination of the protein structure. Further, we believe that the tensors reported in this work will be useful in the cross-correlation and structural studies of proteins using solution NMR spectroscopy. Acknowledgment. We thank Kevin Hallock and Prof. Zuiderweg for helpful discussions. This research was partly supported by the research funds from the College of Literature, Science, and the Arts, and the Horace H. Rackham School of Graduate Studies at the University of Michigan. Acknowledgment is also made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for partial support of this research. Valuable suggestions from the reviewers are gratefully acknowledged. References and Notes (1) Sanders, C. R.; Hare, B. J.; Howard, K. P.; Prestegard, J. Prog. NMR Spectrosc. 1993, 26, 421-444. (2) Cross, T. A.; Opella, S. J. Curr. Opin. Struct. Biol. 1994, 4, 574581. (3) Ramamoorthy, A.; Marassi, F. M.; Opella, S. J. In Proceedings of the International School of Biological Magnetic Resonance, 2nd Course, Dynamics and the Problem of Recognition in Biological Macromolecules; Jardetsky, O., Lefeure, J., Eds.; Plenum: New York, 1996; Chapter 17, pp 237-255. (4) Smith, S. O. Q. ReV. Biophys. 1996, 29, 395-449. (5) Marassi, F. M.; Ramamoorthy, A.; Opella, S. J. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 8551-8556. (6) Ketchem, R. R.; Roux, B.; Cross, T. A. Structure 1997, 5, 16551669.
J. Phys. Chem. B, Vol. 103, No. 39, 1999 8389 (7) Ramamoorthy, A.; Marassi, F. M.; Zasloff, M.; Opella, S. J. J. Biomol. NMR 1995, 6, 329-334. (8) Lee, D. K.; Ramamoorthy, A. J. Magn. Reson. 1998, 133, 204206. (9) Zasloff, M. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 5449-5453. (10) Bechinger, B.; Zasloff, M.; Opella, S. J. Biophys. J. 1992, 62, 1214. (11) Bechinger, B.; Zasloff, M.; Opella, S. J. Protein Sci. 1993, 2, 20772084. (12) Gesell, J.; Zasloff, M.; Opella, S. J. J. Biomol. NMR 1997, 9, 127135. (13) Bennett, A. E.; Rienstra, C. M.; Auger, M.; Lakshmi, K. V.; Griffin, R. G. J. Chem. Phys. 1995, 103, 6951-6958. (14) Lee, M.; Goldburg, W. I. Phys. ReV. 1965, 140A, 1261-1271. (15) Bielecki, A.; Kolbert, A. C.; de Groot, H. J. M.; Griffin, R. G.; Levitt, M. H. AdV. Magn. Reson. 1990, 14, 111-124. (16) Levy, G. C.; Lichter, R. L. Nitrogen-15 Nuclear Magnetic Spectroscopy; John Wiley & Sons: New York, 1979. (17) Lee, D. K.; Wittebort, R. J.; Ramamoorthy, A. J. Am. Chem. Soc. 1998, 120, 8868-8874. (18) Wu, C. H.; Ramamoorthy, A.; Gierasch, L. M.; Opella, S. J. J. Am. Chem. Soc. 1995, 117, 6148-6149. (19) Munowitz, M.; Griffin, R. G.; Bodenhausen, G.; Huang, T. H. J. Am. Chem. Soc. 1980, 103, 2529-2533. (20) Roberts, J. E.; Harbison, G. S.; Munowitz, M. G.; Herzfeld, J.; Griffin, R. G. J. Am. Chem. Soc. 1987, 109, 4163-4169. (21) Nicholson, L. K.; Asakura, T.; Demura, M.; Cross, T. A. Biopolymers 1993, 33, 847-861. (22) Kvick, A.; Al-Karaghouli, A. R.; Koetzle, T. F. Acta Crystallogr. 1977, B33, 3796. (23) Harbison, G. S.; Jelinski, L. W.; Stark, R. E.; Torchia, D. A.; Herzfeld, J.; Griffin, R. G. J. Magn. Reson. 1984, 60, 79-82. (24) Linder, M.; Hohener, A.; Ernst, R. R. J. Chem. Phys. 1980, 73, 4959-4970. (25) Valentine, K. G.; Rockwell, A. L.; Gierasch, L. M.; Opella, S. J. J. Magn. Reson. 1987, 73, 519-523. (26) Oas, T. G.; Hartzell, C. J.; McMahon, T. J.; Drobny, G. P.; Dahlquist, F. W. J. Am. Chem. Soc. 1987, 109, 5956-5962. (27) Oas, T. G.; Hartzell, C. J.; Dahlquist, F. W.; Drobny, G. P. J. Am. Chem. Soc. 1987, 109, 5962-5966. (28) Hartzell, C. J.; Whitfield, M.; Oas, T. G.; Drobny, G. P. J. Am. Chem. Soc. 1987, 109, 5966-5969. (29) Hiyama, Y.; Niu, C. H.; Silverton, J. V.; Bavoso, A.; Torchia, D. A. J. Am. Chem. Soc. 1988, 110, 2378-2383. (30) Teng, Q.; Cross, T. A. J. Magn. Reson. 1989, 85, 439-447. (31) Wasylishen, R. E.; Penner, G. H.; Power, W. P.; Curtis, R. D. J. Chem. Soc. 1989, 111, 6082-6086. (32) Separovic, F.; Smith, R.; Yannoni, C. S.; Cornell, B. A. J. Am. Chem. Soc. 1990, 112, 8324-8328. (33) Curtis, R. D.; Penner, G. H.; Power, W. P.; Wasylishen, R. E. J. Phys. Chem. 1990, 94, 4000-4006. (34) Power, W. P.; Wasylishen, R. E. Annu. Rep. NMR Spectrosc. 1991, 23, 1-84. (35) Mai, W.; Hu, W.; Cross, T. A. Protein Sci. 1993, 2, 532-542. (36) Shoji, A.; Ando, A.; Kuroki, S.; Ando, I.; Webb, G. A. Annu. Rep. NMR Spectrosc. 1993, 26, 55-98. (37) Lumsden, M. D.; Wasylishen, R. E.; Eichele, K.; Schindler, M.; Penner, G. H.; Power, W. P.; Curtis, R. D. J. Am. Chem. Soc. 1994, 116, 1403-1413. (38) Yeo, J. H.; Demura, M.; Asakura, T.; Fujito, T.; Imanari, M.; Nicholson, L. K; Cross, T. A. Solid State Nucl. Magn. Reson. 1994, 3, 209-218. (39) Hong, M.; Gross, J. D.; Griffin, R. G. J. Magn. Reson. 1998, 135, 169-177. (40) Walling, A. E.; Pargas, R. E.; de Dios, A. C. J. Phys. Chem. A 1997, 101, 7299-7303. (41) McConnell, H. M. J. J. Chem. Phys. 1956, 25, 709-711. (42) Mackor, E. L.; Maclean, C. Prog. Nucl. Magn. Reson. Spectrosc. 1967, 3, 129-157. (43) Goldman, M. J. Magn. Reson. 1984, 60, 437-452. (44) Wimperis, S.; Bodenhausen, G. Mol. Phys. 1989, 66, 897-919. (45) Brunschweiler, R.; Ernst, R. R. J. Chem. Phys. 1992, 96, 17581766. Burghardt, I.; Konrat, R.; Bodenhausen, G. Mol. Phys. 1992, 75, 467486. (46) Werbelow, L. G. In Encyclopedia of Nuclear Magnetic Resonance; Grant, D. M., Harris, R. K., Editors-in-Chief; Wiley: London, 1996; Vol. 6, pp 4072-4078. (47) Kumar, A.; Madhu, P. K. Concepts Magn. Reson. 1996, 8, 139160. (48) Rutejans, H.; Kaun, E.; Hull, W. E.; Limbach, H. H. Nuclei Acids Res. 1982, 10, 7027-7039. (49) Gueron, M.; Leroy, J. L.; Griffey, R. J. J. Am. Chem. Soc. 1983, 105, 7262-7266.
8390 J. Phys. Chem. B, Vol. 103, No. 39, 1999 (50) Dalvit, C. J. Magn. Reson. 1992, 97, 645-650. (51) Tjandra, N.; Szabo, A.; Bax, A. J. Am. Chem. Soc. 1996, 118, 6986-6991. (52) Tessari, M.; Vis, H.; Boelens, R.; Kaptein, R.; Vuister, W. J. Am. Chem. Soc. 1997, 119, 8985-8990. (53) Tjandra, N.; Bax, A. J. Am. Chem. Soc. 1997, 119, 9576-9577. (54) Yang, D. W.; Konrat, R.; Kay, L. E. J. Am. Chem. Soc. 1997, 119, 11938-11940. (55) Yang, D. W.; Mittermaier, A.; Mok, Y. K.; Kay, L. E. J. Mol. Biol. 1998, 276, 939-954.
Lee et al. (56) Fischer, M. W. F.; Zeng, L.; Pang, Y. X.; Hu, W. D.; Majumdar, A.; Zuiderweg, E. R. P. J. Am. Chem. Soc. 1997, 119, 12629-12642. (57) Pervushin, K.; Riek, R.; Wider, G.; Wuthrich, K. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 12366-12371. (58) Boyd, J.; Redfield, C. J. Am. Chem. Soc. 1998, 120, 9692-9693. (59) Fushman, D.; Tjandra, N.; Cowburn, D. J. Am. Chem. Soc. 1998, 120, 10947-10952. (60) Fushman, D.; Cowburn, D. J. Biomol. NMR 1999, 13, 139-147. (61) Shoji, A.; Ozaki, T.; Fujito, T.; Deguchi, K.; Ando, I.; Magoshi, J. J. Mol. Struct. 1998, 441, 251-266.
