A Two-Dimensional Magic-Angle Decoupling and Magic-Angle

4752

J. Phys. Chem. B 2001, 105, 4752-4762

A Two-Dimensional Magic-Angle Decoupling and Magic-Angle Turning Solid-State NMR Method: An Application to Study Chemical Shift Tensors from Peptides That Are Nonselectively Labeled with 15N Isotope Dong-Kuk Lee, Yufeng Wei, and A. Ramamoorthy* Biophysics Research DiVision, Department of Chemistry and Macromolecular Science and Engineering, The UniVersity of Michigan, Ann Arbor, Michigan 48109-1055 ReceiVed: August 11, 2000; In Final Form: NoVember 1, 2000

A two-dimensional solid-state NMR technique is presented that can be used to determine the 15N chemical shift and 1H-15N dipolar coupling tensors in powder samples of polypeptides containing 15N isotopes at multiple sites. By combining the magic-angle rf decoupling in one time period and the magic-angle turning pulse sequence in another time period of a 2D experiment, we obtain 2D spectra in which the convoluted chemical shift anisotropy (CSA) and dipolar coupling line shapes appear along one axis and the normal MAS spectrum appears along the other axis. The magnitudes of the principal elements of the 15N CSA tensors and their orientations in the molecular frame for n-acetyl-15N-L-Val-15N-L-Leu (NAVL) and n-acetyl-15N-D,L-Val (NAV) powder samples are determined using this method. The magnitudes of the 15N CSA tensors are 60.2 ( 1, 87.1 ( 1, and 230.1 ( 1 ppm for the Val residue in NAVL; 58.7 ( 1, 93.7 ( 1, and 232.8 ( 1 ppm for the Leu residue in NAVL; 59.6 ( 1, 80.5 ( 1, and 235.3 ( 1 ppm for site I in NAV; and 57.5 ( 2, 81.0 ( 2, and 227.0 ( 2 ppm for site II in NAV. The experimental results also suggest that the most-shielded axis, σ11N, and the σ22N axis of the 15N CSA tensor are significantly tilted away from the peptide plane and the normal to the peptide plane, respectively. The tilt angles (RN) are 34 ( 12° for Val and 36 ( 11° for Leu in NAVL, whereas it is 5 ( 22° in NAV. The angles (βN) between the least-shielded axis of the 15N CSA tensor, σ33N, in the peptide plane and the N-H bond are determined to be 20 ( 2° for Val and 18 ( 2° for Leu in NAVL and 21 ( 2° for NAV. The values are in good agreement with some of the recent solid-state NMR experimental studies on peptides. The values for the βN angle reported in this study are also in agreement with the solution NMR studies on water-soluble proteins.

Introduction The magnitudes and orientations of the chemical shift anisotropy (CSA) and heteronuclear dipolar coupling tensors measured from solid-state NMR experiments provide very powerful information for characterizing rapid, large-amplitude motions in solids1-3 and interpreting the relaxation rates measured with solution NMR spectroscopy.4,5 Characterization of CSA tensors in peptide bonds is also essential for structural studies of membrane-associated proteins using solid-state NMR constraints.6-9 Until recently, solid-state NMR spectroscopy was the only method for experimentally determining the CSA tensors in the molecular frame. Solid-state NMR studies of various polypeptides suggested that the 15N CSA tensor depended on the type of amino acid residue, the secondary structure, the dynamics, and the hydrogen bonding.10-14 Because resolving and assigning resonances from multiple sites of a solid-state sample are difficult, solid-state NMR methods thus far have been limited to the study of peptides that are selectively and site-specifically labeled with 15N. On the other hand, highresolution multidimensional solution NMR methods have recently been used to directly measure the 15N CSA tensors from the backbone of water-soluble proteins uniformly labeled with 15N.15,16 This is feasible, despite CSA averaging due to the fast reorientational molecular motion in solution, because the CSA * Author to whom correspondence should be addressed. Phone: (734)647-6572. E-mail:[email protected].

contributes to the nuclear spin relaxation caused by reorientational motion.5,15,17-24 For example, dipole CSA cross-correlated relaxation experiments probe the correlated modulation of CSA and magnetic heteronuclear dipolar couplings. Fushman et al. demonstrated a method for separating the magnitude and orientation of the 15N CSA tensor in human ubiquitin using relaxation data collected at different magnetic field strengths.15 Further, the influence of the relative orientation of the 15N CSA and 1H-15N dipolar interaction tensors on the relaxation properties of the observed nucleus and on the macromolecular dynamics has been analyzed.16,23 However, conformational exchange contributions, variations in the 1H-15N dipolar couplings, and experimental errors in the measurement of relaxation parameters are some of the possible sources of errors in the determination of CSA tensors using solution NMR methods. Therefore, more accurate CSA tensors determined using solid-state NMR methods are highly valuable in the fitting of motional models using relaxation data. Recently, we demonstrated that a simple one-dimensional dipolar shift method can be used to characterize the 15N chemical shift and 1H-15N dipolar coupling tensors of peptides or proteins that are isotopically labeled with 15N at a single site.25 This method is simple to implement, the results are easy to interpret, and it is less time-consuming and requires less sample than twodimensional SLF experiments.26-32 However, it is not possible to apply this technique to proteins that are labeled with 15N at more than one site because of overlap of the spectral lines from

10.1021/jp002902s CCC: $20.00 © 2001 American Chemical Society Published on Web 05/01/2001

2D Magic-Angle Solid-State NMR Method

J. Phys. Chem. B, Vol. 105, No. 20, 2001 4753

chemically inequivalent 15N sites. This paper describes a twodimensional experiment for measuring the CSA of dilute spins and the heteronuclear dipolar coupling between protons and dilute spins at multiple sites in polycrystalline or amorphous solids. This technique correlates the isotropic chemical shift and the convoluted dipolar shift anisotropic interactions using a combination of magic-angle decoupling33 and magic-angle turning34-39 pulse sequences (MADMAT). This MADMAT method has the advantage of high resolution due to MAS while also retaining the anisotropic dipolar shift interactions associated with the chemically inequivalent sites. Performance of MADMAT under slow-spinning MAS is demonstrated on polycrystalline samples of n-acetyl-L-15N-valyl-L-15N-leucine (NAVL) and n-acetyl-D,L-15N-valine (NAV). Nitrogen-15N CSA and 1H-15N dipolar coupling tensors associated with both of the amide sites in the NAVL dipeptide and with the two inequivalent sites of NAV are determined in this work. Experimental Section Materials. All of the 15N-labeled amino acids were purchased from Cambridge Isotope Laboratory, Andover, MA. NAV and NAVL were synthesized using a procedure explained in the literature.40 Powder samples of NAV and NAVL were recrystallized from water for solid-state NMR experiments. A single crystal was prepared by slow evaporation of a saturated water solution of the respective peptide (NAV or NAVL). Both of the crystals are monoclinic; the crystal structures were confirmed by X-ray diffraction and are in agreement with the data reported in the literature.41 Solid-State NMR Experiments. All of the experiments were carried out using a Chemagnetics Infinity-400 spectrometer. A 5-mm double-resonance MAS probe tuned to 15N and 1H nuclei at frequencies of 40.59 and 400.56 MHz, respectively, was used. The sample spinning rate was stabilized to within (1 Hz using a Chemagnetics MAS controller. A special drive tip was used to stabilize the slow spinning speed of the rotor. Because the MAS controller cannot read spinning speeds below 200 Hz, a 5-mm zirconia rotor was black-marked symmetrically at six different places for experiments with spinning speeds less than 200 Hz. rf field strengths of 98.0 and 55.5 kHz were used for the 90° pulses in the 1H and 15N rf channels, respectively. A 38-kHz rf field was used for cross-polarization (CP) in both of the rf channels. A rf field strength of 98.0 kHz was used for proton decoupling with an offset frequency of 66 kHz to set the magic-angle decoupling condition during the 15N signal acquisition. An experimentally determined scaling factor, 0.56 ( 0.02, for the magic-angle rf irradiation was used in the calculation of the dipolar shift spectra. We take this opportunity to clarify some of the confusion in the literature with regard to the referencing of 15N chemical shift values. Often, solid-state NMR studies make use of polycrystalline samples of (15NH4)2SO4 and 15NH4NO3 under static and MAS conditions to reference the report 15N chemical shift values. On the other hand, solution NMR studies almost always reported the 15N chemical shift values relative to NH3 (liquid, 25 °C) at 0 ppm.42,43 Because the exact values of the 15N CSA tensors are important for the study of biological molecules in both solid and solution phases, we performed experiments on several standard samples to verify the 15N chemical shift values reported in the literature. We assumed the 15N chemical shift of NH3 (liquid, 25 °C) to be zero42 and that of CH3NO3 (liquid) to be 380.2 ppm, as reported in the literature.43 A polycrystalline sample of (15NH4)2SO4 under static conditions at room temperature displayed a single peak at 24.1

Figure 1. Nitrogen-15 chemical shift spectra of a polycrystalline sample of the dipeptide n-acetyl-15N-L-Valyl-15N-L-Leucine under (A) static and (B) MAS conditions. Nitrogen-15 chemical shift spectra of a polycrystalline sample of a model peptide n-acetyl-15N-D,L-Valine under (C) MAS and (D) static conditions. The isotropic 15N chemical shift peaks at 125.7 and 128.4 ppm in (B) are assigned to the Val and Leu residues of NAVL, whereas the peaks at 121.1 and 125.5 ppm are assigned to two different sites of NAV. A 30-mg NAV powder sample and a 25-mg NAVL powder sample were used in the experiments. The spectra in A-D are the result of 256, 16, 12, and 200 scans, respectively

ppm with a line width of 4.8 ppm. On the other hand, under MAS conditions, four peaks at 123.9, 124, 124.2, and 124.3 ppm (with a line width of 0.055 ppm) were observed; the acquisition time, MAS speed, and recycle delay were 512 ms, 5 kHz, and 5s, respectively. This is not in agreement with the literature, which reported only two 15N peaks with a separation of 0.3 ppm in the room-temperature phase at 290 K.44,45 Because there are two pairs of inequivalent ammonium ions in an unit cell,46 only two 15N isotropic chemical shift peaks are expected, as reported by previous studies. However, the four 15N isotropic chemical shift peaks observed in our spectrum could be because of the presence of two different phases in the sample or because all four molecules in the unit cell are magnetically inequivalent. Further, the 15N isotropic chemical shift frequencies of these peaks are temperature-dependent. Therefore, one should be cautious in using the (15NH4)2SO4 powder sample as a standard reference for reporting the 15N isotropic chemical shift values. Some previous studies used a value of 26.8 ppm for the (15NH4)2SO4 powder sample under static conditions. Therefore, it is necessary to convert the 15N CSA values from the literature to a common reference scale before using them. In this paper, all of the 15N spectra are referenced to NH3 (liquid, 25 °C) by setting the observed 15N signal of solid NH4Cl to 38.8 ppm. Results and Discussion The nitrogen-15 chemical shift spectra of a powder sample of NAVL under static and MAS conditions are shown in Figure 1A and B, respectively. There are two magnetically inequivalent molecules per unit cell of an NAVL single crystal,41 and each NAVL molecule consists of two chemically inequivalent 15N sites (valine and leucine residues). Therefore, a maximum of four 15N chemical shift peaks was observed in experiments on a single-crystal sample of NAVL at an arbitrary orientation relative to the direction of the magnetic field (spectrum not

4754 J. Phys. Chem. B, Vol. 105, No. 20, 2001

Lee et al.

Figure 2. (A) Two-dimensional spectrum that correlates the 15N chemical shift anisotropy and isotropic chemical shift of a polycrystalline sample of the dipeptide n-acetyl-15N-L-Valyl-15N-L-Leucine. It was obtained using the triple-echo MAT pulse sequence with a sample rotation rate of 40 ( 1 Hz, an echo delay time ∆ of 20 µs, an acquisition dwell of 60 µs, a t1 increment of 60 µs, 24 scans, and 392 t1 increments. A 2-ppm Gaussian line broadening was applied in both dimensions. (B) and (C) 15N CSA powder patterns obtained from slices of the triple-echo MAT 2D spectrum in A. The simulated spectra are shown as dashed lines in B and C.

shown). The two isotropic 15N chemical shift peaks (125.7 ( 0.5 and 128.4 ( 0.5 ppm) in the CPMAS spectrum of a powder sample of NAVL in Figure 1B are due to the two chemically inequivalent amide sites of the molecule. The 15N CPMAS spectrum of NAVL (n-acetyl-L-15N-valyl-L-leucine) labeled with 15N at a single site consisted of a single peak at 125.7 ppm (spectrum not shown). Therefore, the peaks at 125.7 and 128.4 ppm in the 15N CPMAS spectrum of a uniformly 15N-labeled NAVL powder sample (see Figure 1B) are assigned to the Val and Leu residues, respectively. The 15N CPMAS spectrum of an NAV powder sample, which contains two peaks at 125.5 and 121.1 ppm with an intensity ratio of 10:1, correspond to two different crystalline forms in the sample (see Figure 1C). Further recrystallization of the NAV powder sample from water resulted in a single peak at 125.5 ppm in the 15N CPMAS spectrum. Therefore, the major peak at 125.5 ppm in Figure 1C corresponds to the monoclinic crystalline form of NAV; no efforts were made to determine the other crystalline form of NAV. We designate the peak at 125.5 ppm as site I and the peak at 121.1 ppm as site II throughout this paper. The 15N chemical shift powder pattern spectrum of NAV is given in Figure 1D. This form of the NAV sample was chosen to examine the efficacy of the MAT technique in recovering the CSA tensor for site II, which has a very poor signal-to-noise ratio (see Figure 1C). A difference in the 15N CSA of the two chemically inequivalent amide sites in an NAVL molecule can be expected. However, it is not possible to determine the two overlapping CSA line shapes from a one-dimensional powder spectrum. We assume that the magnitudes of the 15N CSA tensors associated with the same residue in two different, magnetically inequivalent NAVL molecules are the same because their 15N isotropic chemical shifts are identical (see Figure 1B). To obtain the CSA line shapes for the two sites in NAVL and NAV, a twodimensional triple-echo MAT experiment36 was carried out with a spinning frequency of 40 Hz. The details of the twodimensional triple-echo MAT technique and the procedure to process the experimental data can be found elsewhere.36 The two-dimensional MAT spectra correlate the isotropic and

anisotropic 15N chemical shift interactions in NAVL and NAV and are shown in Figures 2A and 3A, respectively. The CSA line shapes for the two chemically inequivalent sites of NAVL are shown in Figure 2B and C and for the two magnetically inequivalent sites of NAV in Figure 3B and C. The spectral lines are well-resolved in both frequency dimensions of the 2D MAT spectra. The isotropic 15N chemical shift frequencies measured from the ω1 dimension of the 2D MAT spectrum in Figure 2A are identical to the values obtained from the CPMAS spectrum in Figure 1B. Because the shoulders of the 15N CSA line shapes obtained from the 2D MAT spectrum are welldefined, the magnitudes of the CSA tensors can be directly measured from the breaks in the powder patterns. The magnitudes (defined as σ33N g σ22N g σ11N) of the 15N CSA tensors were determined by comparing the best-fitting simulated (shown as dashed lines in Figure 2B and C for NAVL and Figure 3B and C for NAV) and experimental spectra (shown as solid lines in Figure 2B and C for NAVL and Figure 3B and C for NAV). In the case of NAVL, the magnitudes of 15N CSA tensors are σ11N ) 60.2 ( 1, σ22N ) 87.1 ( 1, and σ33N ) 230.1 ( 1 ppm for the Val residue and σ11N ) 58.7 ( 1, σ22N ) 93.7 ( 1, and σ33N ) 232.8 ( 1 ppm for the Leu residue. In the NAV molecule, they are σ11N ) 59.6 ( 1, σ22N ) 80.5 ( 1, and σ33N ) 235.3 ( 1 ppm for site I and σ11N ) 57.5 ( 2, σ22N ) 81 ( 2, and σ33N ) 227 ( 2 ppm for site II. The isotropic chemical shift values obtained from averages of the magnitudes of the 15N CSA tensor and directly from the one-dimensional CPMAS spectrum are the same. This suggests that the 15N CSA line shapes obtained from the 2D MAT spectrum and the measured tensor values are accurate. It is obvious that the magnitudes of the 15N chemical shift tensor are different for the two different sites, in particular, the difference in the values of σ22N is significant (6 ppm for NAVL). It is remarkable that the 2D MAT experiment is capable of providing the CSA line shape associated with site II of NAV even though it has a poor signal-tonoise ratio (S/N) (see Figure 3B). Characterization of the 15N CSA tensor along with the 1H15N dipolar coupling tensor is necessary in order to determine the orientation of the principal axes of the CSA tensor in the



Figure 3. (A) Two-dimensional spectrum that correlates the 15N chemical shift anisotropy and isotropic chemical shift of a polycrystalline sample of a model peptide n-acetyl-15N-D,L-Valine. (B) and (C) 15N CSA powder patterns obtained from slices of the triple-echo MAT 2D spectrum in A. The simulated spectra are shown as dashed lines in B and C. All of the experimental conditions are as given in the caption to Figure 2.

Figure 4. (A) Original triple-echo MAT pulse sequence. (B) Two-dimensional pulse sequence that correlates the isotropic 15N chemical shift and dipolar shift interactions. The 90° and 180° pulses are represented by narrow and broad rectangles, respectively. ∆ is the echo delay, and the time T is an integer number of rotor periods (excluding a multiple of three rotor periods). The details on the triple-echo MAT pulses in the t1 period and the phase cycling scheme can be found elsewhere.36 Proton decoupling is achieved by using the TPPM sequence49 during t1 and magic-angle decoupling33 during t2.

molecular frame. Recently, three-dimensional experiments have been developed and shown to be useful in analyzing the chemical shift tensors from multiple sites of a molecule in a powder sample.47,48 Because a three-dimensional experiment is time-consuming and might not be practical for biological samples that are either unstable for such long experiments or not readily available in large quantities, alternate techniques that are comparatively less time-consuming are of great interest. In this paper, a two-dimensional technique (MADMAT) is demonstrated that allows for the determination of the 15N CSA and 1H-15N dipolar coupling tensors of the two chemically inequivalent sites of an NAVL powder sample in the molecular frame. The two-dimensional MADMAT pulse sequence is shown in Figure 4B; the original triple-echo MAT pulse

sequence is given in Figure 4A for comparison. After preparation of the 1H magnetization and cross-polarization (CP) to the 15N nuclei, the 15N magnetization is allowed to evolve under the isotropic chemical shifts using the triple-echo MAT pulse sequence during the period t1,36 while the protons are decoupled using the TPPM decoupling sequence.49 During the acquisition of the 15N magnetization data in the time interval t2, the frequency of the proton decoupling was shifted by an offset in order to establish an effective decoupling field at the magic angle.13 This magic-angle rf irradiation of the protons suppresses the 1H-1H homonuclear dipolar interactions and scales the 1H15N dipolar coupling by a factor 0.58.33 Thus, in the twodimensional spectrum, the ω2 frequency dimension consists of the 15N chemical shift and 1H-15N dipolar interactions modu-


Lee et al.

Figure 5. Two-dimensional MADMAT spectrum that correlates the isotropic 15N chemical shift and 1H-15N dipolar shift interactions of an NAVL powder sample obtained using the pulse sequence in Figure 4B. The spectrum was obtained using 325 t1 experiments with a 60-µs t1 dwell, a spinning speed of 200 ( 1 Hz, an echo delay ∆ of 20 µs, 192 scans, and a 3-s recycle delay. In the t1 period, n was set to 4 rotor periods. A 2-ppm Gaussian line broadening was applied in both the t1 and t2 dimensions. A 15N chemical shift slice taken at 140 ppm is displayed on the chemical shift axis of the 2D spectrum in order to clarify the resolution in the t1 dimension of the technique.

lated by the MAS frequency, whereas the ω1 dimension provides the isotropic 15N chemical shifts. Unlike the original triple-echo MAT sequence shown in Figure 4A, in the MADMAT pulse sequence shown in Figure 4B, an extra time of t1/3 elapses after the echo but before the start of the signal acquisition. This is necessary for several reasons: (1) to retain the 3-fold symmetry of the experiment in the t1 evolution period in order to avoid any poorly phased baseline problems; (2) to avoid the evolution of the 15N magnetization under the scaled 1H-15N dipolar coupling for a time period of t1/3 which otherwise would complicate the spectral frequencies in the ω1 frequency dimension of the twodimensional spectrum; and (3) to obtain a simple twodimensional spectrum that does not require any shearing of the data to measure the isotropic and dipolar shift interactions. The possibility that there could be a sacrifice in the signal-to-noise ratio of the experiment due to the t1/3 delay in signal acquisition was also considered. To examine these points, experiments were performed on the NAVL powder sample using the pulse sequence shown in Figure 4B but with an on-resonance decoupling of protons during signal acquisition in the t2 time interval. The resultant two-dimensional spectrum processed without shearing of the data was identical to the spectrum in Figure 2A and the signal-to-noise ratios were comparable in the two cases. It can be mentioned here that the MADMAT pulse sequence in Figure 4B can be further modified to use other variants of the MAT technique instead of the triple-echo sequence in the t1 period and also other multiple pulse sequences instead of the magicangle rf decoupling in the t2 period. The two-dimensional spectrum of an NAVL powder sample displayed in Figure 5 was obtained using the MADMAT pulse sequence with a spinning speed of 200 Hz. The one-dimensional dipolar shift spectral slices taken at the isotropic 15N chemical shift frequencies (125.7 and 128.4 ppm) of the two-dimensional spectrum in Figure 5 are displayed in Figures 6A and 7A for

the Val and Leu residues, respectively. It is important to note that the 15N CSA line shapes corresponding to the two inequivalent sites in NAVL, with a 2.7-ppm isotropic chemical shift difference, are well-resolved in the 2D MADMAT spectrum. Because the 200-Hz spinning speed is smaller than the resolution of the dipolar shift spectra, spinning sidebands in Figures 6A and 7A are not observed. The signal-to-noise ratio in the high-field region (from ∼20 to 180 ppm in Figure 5) of the dipolar shift frequency dimension is about 5 times larger than that in the low-field region (∼200 to 380 ppm). This is the main reason for the t1 noise in the high-field region of the 2D MADMAT spectrum in Figure 5. This can be realized from a 15N chemical shift slice taken at 140 ppm and displayed on the chemical shift axis of the 2D spectrum in Figure 5. Because the isotropic chemical shift frequency (t1) dimensions in MAT (see Figure 4A) and MADMAT pulse sequences (see Figure 4B) are similar, the resolutions in this dimension of the 2D spectra are expected to be similar (that is about 0.1 ppm).36 The simulated 1D dipolar shift spectra for the slices taken at the isotropic 15N chemical shift frequencies (125.7 and 128.4 ppm) of the two-dimensional spectrum are shown in Figures 6B and 7B for the Val and Leu residues, respectively, in NAVL. The magnitudes of the 15N CSA tensor measured from Figure 2B and C were used in the simulations. In the simulations, the angle RN (the angle between the projection of the N-H bond on the peptide plane and the most-shielded axis, σ11N, of the 15N CSA tensor) was varied from 0 to 90°; β (the angle N between the least-shielded axis, σ33N, and the N-H bond in the peptide plane) was varied from 10 to 30°; and a N-H bond length of 1.06 Å was used. The details on the simulation of a dipolar shift spectrum are given in our previous publication.25 The root-mean-square deviations (RMSD) between the simulated and experimental spectra were calculated in order to estimate the error in the measured RN and βN angles. The 2D RMSD contour plots are presented in Figures 6C and 7C. The



Figure 6. (A) Dipolar shift powder pattern for the Val residue obtained from the slices at 125.7 ppm of the 2D spectrum in Figure 5. (B) Bestfitting simulated spectrum obtained using RN ) 34 ( 12° and βN ) 20 ( 2°. (C) RMSD contour plot for RN varying from 0 to 90° and βN varying from 10 to 30°. Contour height ranges from 0 to 100% of the highest RMSD value at 5% interval. The estimated errors for RN and βN are obtained at the 5% contour level.

Figure 7. (A) Dipolar shift powder pattern for the Leu residue obtained from the slices at 128.4 ppm of the 2D spectrum in Figure 5. (B) Bestfitting simulated spectrum obtained using RN ) 36 ( 11° and βN ) 18 ( 2°. (C) RMSD contour plot for RN varying from 0 to 90° and βN varying from 10 to 30°. Contour height ranges from 0 to 100% of the highest RMSD value at 5% interval. The estimated errors for RN and βN are obtained at the 5% contour level.

RMSD between experimental and simulated spectra is defined as

RMSD(RN, βN) )

{

1

N

∑

N - 1 i)1

[Siexp

-

}

1/2

Sisim(RN,βN)]

where N is the number of data points and Sexp and Ssim are the intensities of the experimental and simulated spectra, respectively. Contour heights range from 0 to 100% from the lowest to the highest RMSD values at 5% interval. The estimated errors for RN and βN are obtained at the 5% contour level. The bestfitting spectrum in Figure 6B was obtained using RN ) 34 ( 12° and βN ) 20 ( 2° for the Val reside. On the other hand, the best-fitting spectrum in Figure 7B was obtained using RN ) 36 ( 11° and βN ) 18 ( 2° for the Leu reside. 2D MADMAT experiments were also carried out with various spinning frequencies ranging from 40 to 500 Hz. At a higher spinning speed, the isotropic chemical shift evolution time was set to nτr, where n is any integer except a multiple of 3 and τr is one rotor period of MAS. For example, n)1, 4, and 10 were

used for the spinning speeds 40, 200, and 500 Hz, respectively. The S/N was significantly improved in experiments with a higher spinning speed, and consequently, the experimental time was considerably reduced for fast spinning conditions. The 2D MADMAT spectrum of NAVL powder sample obtained with a spinning speed of 500 Hz is shown in Figure 8. The t1 noise in the high-field region of the 500-Hz 2D spectrum (see Figure 8) is less than that of the 200-Hz 2D spectrum (see Figure 5). This can be realized by comparing the 15N chemical shift slices taken at 140 ppm and displayed on the chemical shift axes of the 2D spectra in Figures 5 and 8. The one-dimensional dipolar shift spectral slices taken at the isotropic 15N chemical shift frequencies (125.7 and 128.4 ppm) of the two-dimensional spectrum in Figure 8 are displayed in Figures 9A and 10A for the Val and Leu residues, respectively. The corresponding simulated spectra are shown in Figures 9B and 10B, and the corresponding 2D RMSD contour plots to estimate the errors in RN and βN are presented in Figures 9C and 10C. The bestfitting spectrum in Figure 9B was obtained using RN ) 44 ( 13° and βN ) 19 ( 2° for the Val reside. On the other hand,


Lee et al.

Figure 8. Two-dimensional spectrum that correlates the isotropic 15N chemical shift and 1H-15N dipolar shift interactions of an NAVL powder sample obtained using the pulse sequence in Figure 4B. The spectrum was obtained using 326 t1 experiments with a 60-µs t1 dwell, a spinning speed of 500 ( 1 Hz, an echo delay ∆ οφ 20 µs, 144 scans, and a 3-s recycle delay. In the t1 period, n was set to 10 rotor periods. A 2-ppm Gaussian line broadening was applied in both the t1 and t2 dimensions. A 15N chemical shift slice taken at 140 ppm is displayed on the chemical shift axis of the 2D spectrum in order to clarify the resolution in the t1 dimension of the technique.

Figure 9. (A) Dipolar shift powder pattern for the Val residue obtained from the slices at 125.7 ppm of the 2D spectrum in Figure 8. (B) Bestfitting simulated spectrum obtained using RN ) 44 ( 13° and βN ) 19 ( 2°. (C) RMSD contour plot for RN varying from 0 to 90° and βN varying from 10 to 30°. Contour height ranges from 0 to 100% of the highest RMSD value at 5% interval. The estimated errors for RN and βN are obtained at the 5% contour level.

the best-fitting spectrum in Figure 10B was obtained using RN ) 41 ( 11° and βN ) 17 ( 3° for the Leu reside. These values, within experimental errors, are in good agreement with the results obtained from 2D MADMAT experiments with various other spinning speeds. The poor signal-to-noise ratio in the low-field region of the dipolar shift slices in Figures 9 and 10 could be a major source of estimated errors in the reported values of RN and βN. A one-dimensional dipolar shift experiment under 500-Hz MAS performed on a single-site-labeled peptide sample also resulted in a poor signal-to-noise ratio in the lowfield region of the spectrum (data not provided). However, the

signal-to-noise ratio in the low-field region of the dipolar shift depended on the number of scans used in the experiment; it can also be improved at the expense of resolution of the spinning sidebands in the high-field region of the spectrum. This suggests that the qualities of the dipolar shift spectra obtained from the 2D MADMAT and 1D dipolar shift techniques are comparable. Because there are two inequivalent sites in the NAV sample (see Figure 1C), the two-dimensional MADMAT experiment was performed to determine the 15N CSA tensors in the molecular frame (spectra not shown). The 15N CSA tensor for site I, with an isotropic chemical shift 125.5 ppm, is given by



Figure 10. (A) Dipolar shift powder pattern for the Leu residue obtained from the slices at 128.4 ppm of the 2D spectrum in Figure 5. (B) Best-fitting simulated spectrum obtained using RN ) 41 ( 11° and βN ) 17 ( 3°. (C) RMSD contour plot for RN varying from 0 to 90° and βN varying from 10 to 30°. Contour height ranges from 0 to 100% of the highest RMSD value at 5% interval. The estimated errors for RN and βN are obtained at the 5% contour level.

TABLE 1: Nitrogen-15 Chemical Shift Tensorsa of the Valine Residue sample NAVe

NAVL:f 200-Hz MAS 500-Hz MAS Val26 in ubiquitin gramicidine Ag Val1 Val6 Val7 Val8(dry) Val8(hydrated and at 143 K) Boc-[1-13C]Gly[15N]ValGlyAla-OPac L-Val in polyalanine L-Val in polyleucine poly(L-Val)-Ih poly(L-Val)-IIh

RN (°)

Rc/104

∆σd

conformation

ref

21 ( 2 19 ( 2 20 ( 2 19 ( 2 -

20 ( 15 0 -25 34 ( 12 44 ( 13 -

1.18 1.04 1.21 1.07 1.06 1.06 -

165.1 157.8 167.7 156.5 156.0 156.0 -

i i 13 51 52 i i 53 54, 55

125.6 119.6 120.6 115.6 121.6

13.6 ( 2 14.5 ( 2 15.5 14.5 14.5 ( 2

28 ( 5 0(5 0 0 0 ( 10

(site I) (site II) right-handed β-helix

1.15 1.04 1.04 1.10 1.04

162.0 152.5 154.0 159.5 154.5

218.8

119.8

10.5

0

0.97

148.0

-

119.1 118.1 126.5 127.5

-

-

-

σ11N

σ22N

σ33N

σisob

59.6 57.5 59.6 59.1 60.2 60.2 -

80.5 81.0 80.0 84.1 87.1 87.1 -

235.3 227.0 238.0 228.1 230.1 230.1 -

125.5 121.1 125.5 123.8 125.7 125.7 123.6

59.6 57.6 57.6 48.6 58.6

83.6 82.6 80.6 75.6 81.6

233.6 222.6 223.6 221.6 224.6

61.8

79.8

-

-

βN (°)

-

1 -

56

R-helix R-helix β-sheet β-sheet

57 57 50 57

All of the chemical shift values are reported relative to liquid NH3 at 0.0 ppm (at 25 °C). b All of the σiso values were measured from CPMAS experiments. c R ) (σ33N - σiso)2/(1 + η2/3) with η ) (σ22N - σ11N)/(σ33N - σiso). d ∆σ ) σ33N - 0.5(σ22N + σ11N). e NAV ) N-acetyl-D,L-valine. f NAVL ) N-acetyl-L-valyl-L-leucine. g β values for the gramicidine A sample were originally reported as the angle H-N-C , whereas they are N 1 reported here as the angle between the N-H bond and σ33N by assuming that the angle H-N-C1 ) 119.5° and that the N-H bond and σ33N lie in the same plane. h Poly(L-Val)-I has a degree of polymerization (DP) ) 100, and poly(L-Val)-II has DP ) 50. i Present work. a

the parameters σ11N ) 59.6 ( 1 ppm, σ22N ) 80.5 ( 1 ppm, σ33N ) 235.3 ( 1 ppm, RN ) 5 ( 22°, and βN ) 21 ( 2°. In the previous study using the one-dimensional dipolar shift technique with no sample spinning, we reported the 15N CSA tensor (σ11N ) 59.6 ppm, σ22N ) 80.0 ppm, σ33N ) 238.0 ppm, RN ) 0°, and βN ) 19°) for the same NAV sample.13 The difference in the results can be attributed to the error in the earlier study due to the overlap of 15N CSA tensors associated with sites I and II. Because of the poor S/N from site II of the NAV sample, we could not determine the orientation of the CSA tensor associated with site II. Nevertheless we have measured the magnitudes of the CSA tensor for this site of the NAV powder sample.

The 15N CSA tensors associated with the amide site of the valine residue in NAV and NAVL are similar (see Table 1). Nitrogen-15 CSA tensors of the Val1,13,50-57 and Leu1,50,55,57-61 amino acid residues from various polypeptides reported in the solid-state NMR literature are summarized in Tables 1 and 2. The variation in the values of σ11N, σ22N, σ33N, σiso (isotropic chemical shift), and ∆σ (chemical shift span) are, respectively, 8.3, 13.5, 13.4, 9.5, and 16.7 ppm for the Val residue and 8.8, 21.6, 17.2, 11.6, and 21 ppm for the Leu residue. The average values of σ11N, σ22N, σ33N, σiso, and ∆σ are 58.8, 81.0, 223.2, 122.2, and 151.2 ppm for Leu and 58.8, 82.0, 227.0, 122.6, and 156.5 ppm for Val in polypeptides. On the other hand, from the studies using solution NMR spectroscopy of proteins, the


Lee et al.

TABLE 2: Nitrogen-15 Chemical Shift Tensorsa of the Leucine Residue βN (°)

RN (°)

Rb/104

∆σc

128.4 128.4

18 ( 2 17 ( 3

36 ( 11 41 ( 11

1.04 1.04

156.5 156.5

224.4

117.4

20 ( 3

30 ( 15

1.13

159.8

221.41

117.0

20 ( 3

30 ( 15

1.06

156.0

-

k

74.3

214.3

116.3

17

10-30

0.95

146.4

-

58 59

σ11N

σ22N

σ33N

NAVL:d

58.7 58.7

93.7 93.7

232.8 232.8

[15N]leu-19 pardaxin

52.3

76.9

[15N]leu-10 SA peptide

51.0

79.9

Ala-Leu

61.3

sample 200-Hz MAS 500-Hz MAS

σiso

conformation -

ref j j k

Met-Leu-Phe

-

-

-

118

-

-

-

-

-

enkephalin I at 296 K I at 273 K II at 273 K III at 296 K

-

-

-

128.0 126.5/129.9 124.6 124.3/131.1

-

-

-

-

β-bend form L-shaped form Extended form

gramicidin Ae D-Leu4 (dry) D-Leu4 (hydrated and at 143 K) D-Leu10 (dry) D-Leu12 (dry) D-Leu12 (hydrated and at 276 K) D-Leu12 (hydrated and Na+ at 276 K) D-Leu14 (dry) Leu14 (hydrated and at 143K)

53.6 62.3 58.6 58.6 63.6 65.6 55.6 64.6

84.6 84.3 88.6 86.6 89.6 89.6 81.6 84.6

219.6 234.6 224.6 216.6 215.6 217.6 218.6 228.6

119.6 127.6 123.9 120.6 122.9 124.3 118.6 125.6

14.5 14.5 14.5 14.5 14.5 14.5 14.5 14.5

0 0 0 0 0 0 0 0

0.97 1.13 0.98 0.90 0.84 0.85 0.98 1.05

[*Leu-Leu-Val]nf,g + 20 0 80 + 20 10 70 + 20 30 50 + 20 50 30 + 20 60 20

-

85.7 85.9 75.9 74.6 74.6

-

127.6 127.6 117.6 117.5 117.4

-

-

-

-

β-sheet β-sheet R-helix R-helix R-helix

[*Leu-Leu-Ile]n + 20 0 80 + 20 30 50

-

78.6 76.6

-

127.3 117.7

-

-

-

-

β-sheet R-helix

[*Leu-Leu-Ala]n + 20 0 80 + 20 30 50 + 20 60 20

-

76.1 76.1 71.6

-

116.8 117.1 117.4

-

-

-

-

R-helix R-helix R-helix

[*Leu-Leu-Gly]n + 20 0 80 + 20 30 50 + 20 60 20

-

81.8 76.1 73.8

-

117.5 117.8 117.4

-

-

-

-

β-sheet R-helix R-helix

[*Leu-Lys(Z)]n + 20 80

-

83.6

-

125.7

-

-

-

-

β-sheet

57

77.3

-

118.3

-

-

-

-

R-helix

57

R-helix -

-

-

-

57

118.2

-

-

β-sheet RR-helix

57 50

-

-

β-sheet

50

[*Leu-Leu-Asp]n + 20 0 80

-

[*Leu-Leu-Glu]n + 20 30 50

-

[Leu]n-Ih [Leu]n-IIh

-

87.5 76.3

-

127.6 117.6

-

-

Z-(L-Leu)6OEti

-

-

-

127.6

-

-

150.5 161.3 151.0 144.0 139.0 140.0 150.0 154.0

70.7

60

right-handed β-helix

55 1 55 55 55, 61 55, 61 55 1, 55 57

57

57

57

All of the chemical shift values are reported relative to liquid NH3 at 0.0 ppm (at 25 °C). R ) (σ33N + with η ) (σ22N σ11N)/(σ33N - σiso). c ∆σ ) σ33N - 0.5(σ22N + σ11N). d NAVL: N-acetyl-L-valyl-L-leucine. e βN values for the gramicidin A sample were originally reported as the angle H-N-C1, whereas they are reported here as the angle between the N-H bond and σ33N by assuming the angle H-N-C1 ) 119.5oand N-H bond and σ33N lie in the same plane. f + indicates the percentage of the respective amino acid residue in the polypeptide. g * indicates the residue labeled with 15N. h [Leu]n-I with a degree of polymerization (DP) ) 5, and [Leu]n-II with DP ) 50. i Z ) benzyloxycarbonyl. j Present work. k Manuscript in preparation. a

average value of σiso is 120.8 ppm for Val and 123.3 ppm for Leu.62 Studies have shown that the σ22N and σiso values are highly sensitive to the backbone conformation of the polypeptide in both the solid63,64 and the solution62 states. Further, results from solid-state NMR and ab initio studies have predicted that σ22N and σ33N are related to the hydrogen-bond length (RN‚‚‚O) and the hydrogen-bond angle (the angle between the N-H and H‚‚‚O bonds), whereas σ11N increases with increasing RN‚‚‚O for the glycine residue.57 However, this trend might not be valid for other residues, as shown here for Val and Leu. For example, RN‚‚‚O ) 2.867 Å41 and σ11N ) 58.7 ppm for Leu of NAVL, RN‚‚‚O ) 2.885 Å41 and σ11N ) 60.2 ppm for Val of NAVL,

b

σiso)2/(1

η2/3)

and RN...O ) 3.184 Å41 and σ11N ) 59.6 ppm for NAV. Therefore, more experimental 15N CSA tensors, as well as ab initio calculations, are essential to understand the variation of 15N CSA tensors associated with residues other than glycine. The variation in the RN and βN angles is not known as there are only a few studies that report the orientation of the 15N tensor in the molecular frame. The values of the βN angle reported in this paper are in good agreement with the results of previous solid-state NMR studies on peptides in the literature,12,25,52,55,58,63,65-69 whereas the values of the RN angle are in good agreement with some of the recent reports in the literature.12,25,52,58,63 It is interesting to note that the value of

2D Magic-Angle Solid-State NMR Method the βN angle determined in this study is also in good agreement with the results of solution NMR studies on water-soluble proteins, which reported βN values in the range of 8-21°.15,70 The chemical shift span, ∆σ, of the 15N CSA tensors reported from solution NMR studies on water-soluble proteins varies from 133 to 180 ppm for the Val residue and from 138 to 193 ppm for the Leu residue.15,16 On the other hand, solid-state NMR studies predict a 140-173 ppm range for the Val residue (see Table 1) and a 130-172 ppm range for the Leu residue (see Table 2). Our results demonstrate that the MADMAT pulse sequence can be used to characterize the CSA and dipolar coupling tensors from multiple sites of a molecule. The 2D MAT technique can be used to obtain magnitudes of CSA tensors from powder samples of protein that consist of 15N isotopes in many sites as long as the isotropic chemical shift difference is larger than 0.1 ppm. The 2D MADMAT technique can be used to determine the RN and βN angles in order to define the orientation of the 15N CSA tensor in the molecular frame. Unfortunately, the key problem hampering widespread use of solid-state NMR spectroscopy for the structure determination of membrane-associated peptides and proteins is the lack of a comprehensive resonance assignment protocol for studying samples that are uniformly or nonselectively labeled with a specific isotope (for example, 15N, 13C, etc.). In this context, the recently developed resonance assignment strategies are encouraging.53,59 Conclusion The experimental results described above demonstrate that the two-dimensional MADMAT technique provides a means of obtaining resolved dipolar shift powder patterns that have the same line shapes as those of the corresponding stationary patterns. The 15N CSA tensors from NAVL and NAV samples have been characterized in the molecular frame using the 2D MAT and 2D MADMAT sequences. It is important to note that the 2D MADMAT experiment can also be used with higher spinning speeds (up to ∼1.5 kHz). The 15N chemical shift and 1H-15N dipolar coupling tensors reported in this paper are in good agreement with the results of some of the recent solidstate NMR studies on peptides, as well as with the results from quantum chemical calculations. The chemical shift span, ∆σ, of the 15N CSA tensors reported from solution NMR studies on water-soluble proteins are in agreement with the solid-state NMR studies. It is interesting to note that the value of the βN angle determined in this study also is in good agreement with the results of solution NMR studies on water-soluble proteins, which reported βN values in the range of 8-21°.15,70 We expect that this technique will be useful in determining the orientation of molecular fragments, the molecular geometry, and the anisotropic intramolecular dynamics in solid as well as solution states. The 15N CSA tensors reported in this paper will be valuable in the cross-correlation studies on proteins using solution NMR methods and in the structure determination of membrane-associated proteins using solid-state NMR methods. Acknowledgment. This research was partly supported by NSF through Grant MCB-9875756 (CAREER Development Award to A.R.). We thank Dr. Cindy Ridenhour (Chemagnetics/ Varian) for help with the MAT experiments. References and Notes (1) Lazo, N. D.; Hu, W.; Cross, T. A. J. Magn. Reson. B 1995, 107, 43-50. (2) Naito, A.; Fukutani, A.; Uitdehaag, M.; Tuzi, S.; Saito, H. J. Mol. Struct. 1998, 441, 231-241.

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A Two-Dimensional Magic-Angle Decoupling and Magic-Angle

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