- Email: [email protected]
Broadband-PISEMA solid-state NMR spectroscopy
Chemical Physics Letters 407 (2005) 289–293 www.elsevier.com/locate/cplett
Broadband-PISEMA solid-state NMR spectroscopy K. Yamamoto, D.K. Lee, A. Ramamoorthy
*
Biophysics Research Division and Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA Received 22 February 2005; in final form 18 March 2005 Available online 13 April 2005
Abstract The measurement of heteronuclear dipolar couplings using a 2D separated-local-field technique, Polarization Inversion Spin Exchange at the Magic Angle (PISEMA), is a unique way to determine the structure, dynamics and topology of molecules in solid-state. However, the resolution and sensitivity of PISEMA are highly dependent on the offset frequency of protons. To overcome this difficulty, in this study, a broadband-PISEMA pulse sequence is proposed. Experimental data from a single crystal and simulated results suggest that the new sequence compensates the offset effects. This is accomplished using a pair of 180° pulses that invert the spin-locked magnetization of I and S nuclei after certain number of SEMA cycles in the t1 period. In addition, unlike PISEMA, BB-PISEMA provides offset-independent dipolar coupling line shapes even when low rf fields are applied. Ó 2005 Elsevier B.V. All rights reserved.
1. Introduction Polarization Inversion Spin Exchange at the Magic Angle (PISEMA) is a 2D separated-local-field (SLF) experiment that correlates the heteronuclear dipolar coupling with the chemical shift of low c nuclei [1–4]. The unique advantage of this method over other types of SLF experiments is that it has a large scaling factor (0.816; that is 18.4% of the heteronuclear dipolar coupling is suppressed by the pulse sequence in the t1 period) and provides very narrow dipolar coupling spectral lines. Therefore, this technique is commonly used to determine the backbone conformation and topology of aligned molecules such as membrane- associated peptides and proteins [4–6]. Recent studies have extended the application of this technique to studies under magic angle spinning (MAS) [2,4] and also to characterize liquid crystalline materials [7]. The process SEMA has also been utilized in the development of new solid-state NMR methods [4,8]. However, the major disadvantage of PISEMA is its dependency on 1H offset [4]. Ramped *
Corresponding author. Fax: +1 734 763 2307. E-mail address: [email protected] (A. Ramamoorthy).
0009-2614/$ - see front matter Ó 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2005.03.082
spin-lock of low c nuclei during SEMA [9] and a new pulse sequence, SAMMY [10], are shown to be relatively less sensitive to offset effects of protons. Another way to overcome offset effects is to use very high power rf pulses. However, use of high rf power is technically demanding and it is not desirable for biological samples as they are sensitive to heat. These problems are severe for solid-state NMR studies at high magnetic fields. Recent studies have demonstrated the use of time averaged nutation (TAN) concept to reduce the power requirement in PISEMA applications [11,12]. In this study, we demonstrate a modified broadband-PISEMA (BBPISEMA) sequence to compensate the offset and other pulse imperfection effects.
2. Results and discussion Spin-locking 1H magnetization at the magic angle in the SEMA part (i.e., the t1 period) of the PISEMA pulse sequence (Fig. 1a) results in the magnetization exchange via the heteronuclear dipolar coupling that is scaled by sinhm (where hm is the magic angle). On the other hand, the effective field direction of the flip-flop Lee–Goldburg
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K. Yamamoto et al. / Chemical Physics Letters 407 (2005) 289–293
Fig. 1. Pulse sequences for spin exchange at magic angle: (a) the original PISEMA sequence; (b) a broadband SEMA (or BB-SEMA) sequence that can be used in the t1 period of the PISEMA sequence to compensate the offset effects. The p pulses in the sequence can be separated by several cycles of FFLG (flip-flop Lee–Goldburg) depending on the required spectral width in the dipolar coupling dimension.
(FFLG) sequence depends on the offset frequency, and therefore, the degree of 1H–1H dipolar decoupling, the efficiency of the 1H spin-lock, the scaling factor, and the rate of spin exchange under the SEMA sequence depend on the 1H offset value as revealed by the previous studies [4,9]. For example, 1H offset increases the extent of spin-locked magnetization that do not participate in the spin exchange process during SEMA which reduces the signal intensity by increasing the zero frequency peak intensity in the dipolar coupling spectrum. It also increases the scaling factor and the line width [4,10]. The dephasing of the 1H magnetization from the magic angle direction due to offset effects during the FFLG sequence of SEMA can be eliminated by simultaneously inverting the direction of the spin-locked magnetization of both I and S nuclei as shown in Fig. 1b. Therefore, it is desirable to have two p pulses for each dwell time in the t1 dimension of the PISEMA experiment as shown in Fig. 1. Since a p pulse is also ap-
plied in the S spin channel, the BB-SEMA sequence should also be less sensitive to S spin offset and effects due to rf field inhomogeneity. All experiments were performed on a Chemagnetics/ Varian Infinity 400 MHz spectrometer using a 5 mm triple-resonance MAS probe at room temperature. An rf field strength of 50 kHz at 35.355 kHz offset was used to set-up the Lee–Goldburg condition in the 1H channel with an effective field strength of 61.2 kHz. An rf field strength of 61.2 kHz was used to spin-lock the 15N magnetization during SEMA and 75 kHz was used to decouple protons using the TPPM sequence [13] during 15N signal acquisition. Since there are two magnetically non-equivalent sites in an unit cell of NAVL (n-acetyl15 15 15 L- N-valyl-L- N-leucine) crystal, the N chemical shift spectrum (data not shown) consists of four well resolved lines from two different amide–15N sites of the dipeptide. 2D experiments were performed for various offset values of 1H and 15N nuclei in order to compare the efficiency
K. Yamamoto et al. / Chemical Physics Letters 407 (2005) 289–293
291
Fig. 2. 2D correlation of 15N chemical shift and 1H–15N dipolar coupling of n-acetyl-L-15N-valyl-L-15N-leucine single crystal at an arbitrary orientation with respective to the external magnetic field: (a) PISEMA and (b) BB-PISEMA spectra. The 2D PISEMA spectrum: (a) was obtained from 128 t1 increments with a dwell time of 32.66 ls (4.18 ms acquisition) while the BB-PISEMA spectrum; (b) was the resultant of 48 t1 increments with a dwell time of 40.86 ls (1.961 ms acquisition). Other experimental parameters include a 500 ls contact time for ramp-CP, four scans and a recycle delay of 3 s.
of the regular PISEMA and the BB-PISEMA pulse sequences. Sample 2D spectra for an arbitrary orientation of the crystal are given in Fig. 2. (a) Experimental
(b) Simulations Dipolar Coupling ( kHz)
18
Experimentally measured (Fig. 3a) and simulated (Fig. 3b) dipolar coupling values as a function of 1H offset in PISEMA and BB-PISEMA sequences are given in
14
PISEMA 25
1H-15N
10
35
BB-PISEMA
15 6
-30
0
-30
-40
-20
0
20
40
Percentage of 1H Offset during t 1
Scaling Factor of BB-SEMA
(c) Simulations 0.8
0.7
0.6
0
10
20
π pulse power/power of LG
Fig. 3. (a) Experimentally measured and (b) Simulated 1H–15N dipolar coupling values as a function of 1H offset. The experimental values were measured from 2D PISEMA (open circles and open rectangles) and BB-PISEMA (filled circles and filled rectangles) spectra of NAVL single crystal obtained under the same experimental conditions. The solid line and dashed lines in (a) belong to two different 15N chemical shift resonances at 83.7 and 102.2 ppm of the spectrum (Fig. 2), respectively. The solid line and the dashed–dotted lines in (b) were simulated for 100 and 75 kHz rf field strengths. (c) Variation of the scaling factor with the ratio of the rf power used for the p and LG pulses in the BB-PISEMA. In the simulations, 100 kHz rf field was used for LG and the p pulse power was varied.
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K. Yamamoto et al. / Chemical Physics Letters 407 (2005) 289–293
Fig. 3. The power of the p pulse used in the experiment and the simulation was the same as the spin-lock rf power in the respective rf channels. On-resonance scaling factors were 0.816 for PISEMA and 0.73 for BB-PISEMA. The percentage of 1H offset in Fig. 3 is defined as (d/LG)*100; where d is the deliberately set offset of the 1H rf carrier and LG is the offset frequency needed to set the effective field direction of SEMA at the magic angle. For example, 10% offset in Fig. 3 is equal to 3.5355 kHz when LG = 35.355 kHz and mrf = 50 kHz were used. The dipolar couplings measured from the 2D PISEMA spectrum are highly dependent on the 1H offset value (shown in open circles and open rectangles). Even a 10% offset increases the dipolar splitting by 20% of the on-resonance dipolar splitting in the PISEMA spectrum (Fig. 3a). This is because the scaling factor of the FFLG and hence the SEMA sequence change with the 1H offset value [4,14–17]. In fact, at large 1H offset values, the dipolar coupling was not measurable as there was no observable signal in the PISEMA spectrum. On the other hand, as shown in Fig. 3, the introduction of a p pulse (Fig. 1b) suppresses the offset effects (shown in filled circles and filled rectangles). For example, 30% offset in the BB-PISEMA sequence changes the dipolar splitting by 5% of the on-resonance dipolar splitting (Fig. 3a). The offset effects on PISEMA and BB-PISEMA were simulated using SIMPSON [18]. A 1H and 15N spin pair with a dipolar coupling constant of 10 kHz was considered in the simulations. The simulated spectra (not shown) for increasing 1H offset values showed an increase in the intensity of the zero-frequency peak while the intensity of the lines separated by the dipolar coupling value decreased. This is because the increase in the offset field increases the magnetization spinlocked along an axis that is away from the magic angle direction. Since this spin-locked magnetization does not participate in the spin exchange process, it results in a strong zero-frequency peak in the x1 dimension of the 2D PISEMA spectrum. As seen from Fig. 3b, simulated results are in good agreement with the experimental data even though the homonuclear dipolar couplings among protons were not included in the simulations. Two different rf field strengths (75 and 100 kHz) were used in the simulations to test the rf power dependency of these pulse sequences. Results suggest that the efficiency of the BB-PISEMA is independent of the rf power used for as much as 35% offset. On the other hand, the efficiency of the PISEMA is better when a higher rf power was used and becomes poor as the rf power was reduced. The scaling factor of the BB-PISEMA depends on the rf power of the p pulse and also on the ratio of the duration of FFLG and p pulse width. As shown in Fig. 3c, it approaches the scaling factor of the PISEMA sequence (0.8165) as the power of the p pulse is increased. Inter-
estingly, the scaling factor of BB-PISEMA is 0.73 even for a moderate p pulse length of 8.2 ls. Sample dipolar coupling slices for two different 1H offset values are given in Fig. 4. The line width for onresonance PISEMA appears to be better than that of BB-PISEMA as longer t1 acquisition was used for PISEMA experiments; whereas experiments with similar t1 acquisition resulted in comparable line widths. However, as shown in Fig. 5, the shape and resolution of PISEMA spectral lines highly depend on the 1H offset value. The offset effect is even stronger when the 1 H–15N dipolar splitting is smaller. For example, the dipolar coupling slices corresponding to other two sites (at 137.2 and 167.8 ppm) that have smaller dipolar coupling values (Fig. 2) are in the noise level even for an offset of ±2 kHz. On the other hand, as shown in Fig. 4, the dipolar coupling spectra measured using the BB-PISEMA sequence are insensitive to the offset effects. Experiments were also carried out to examine the role of 15N offset and the results are given in Fig. 5. PISEMA is less sensitive to 15N offset as compared to 1H offset at least when strong rf fields are used. Our experimental results suggest that the BB-PISEMA sequence (shown in filled circles and filled squares in Fig. 5) is more tolerant to 15N offset effects than the regular PISEMA sequence (shown in empty circles and empty squares in Fig. 5). PISEMA data are not shown for large 15N offset values as the dipolar couplings were not observable in the PISEMA spectra. These results demonstrate that the new sequence, BB-PISEMA, compensates the offset effects of both the dipolar coupled nuclei. This is
Fig. 4. 1H–15N dipolar coupling spectra obtained at different 1H offset values from PISEMA (A) and BB-PISEMA (B). Sites A and B correspond to a 15N chemical shift value of 83.7 and 102.2 ppm, respectively.
K. Yamamoto et al. / Chemical Physics Letters 407 (2005) 289–293
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of the sequence can be improved by reducing the p pulse width. We believe that the BB-PISEMA sequence will be highly valuable in the structural studies of biological molecules such as membrane-associated peptides and proteins and also liquid crystalline materials.
10
Acknowledgement
Dipolar Coupling ( kHz)
14
1H- 15N
293
This research was supported by the research funds from NIH (AI054515 to A.R).
8
References 6
-20
-10 15N
0
10
20
Offset (kHz) during t 1
Fig. 5. Variation of experimentally measured 1H–15N dipolar coupling values as a function of 15N offset in PISEMA (dashed lines) and BBPISEMA (solid lines) sequences. Open (PISEMA) and filled (BBPISEMA) circles correspond to the Site A at 83.7 ppm, and open (PISEMA) and filled (BB-PISEMA) squares correspond to the Site B at 102.2 ppm.
significant for experiments under low rf power conditions and/or at high magnetic fields.
3. Conclusion We have demonstrated that the newly designed pulse sequence, BB-PISEMA, is capable of compensating offset effects of both the heteronuclei. The dipolar splitting and the line shape of BB-PISEMA spectra do not depend on offset values for a bandwidth of 10 kHz when a 50 kHz rf field was used and this bandwidth increases with an increase in the rf power. The scaling factor of this sequence is identical to PISEMA when an ideal pulse is used while it is
[1] C.H. Wu, A. Ramamoorthy, S.J. Opella, J. Magn. Reson. A 109 (1994) 270. [2] A. Ramamoorthy, S.J. Opella, Solid State NMR Spectrosc. 4 (1995) 387. [3] A. Ramamoorthy, C.H. Wu, S.J. Opella, J. Magn. Reson. 140 (1999) 131. [4] A. Ramamoorthy, Y. Wei, D.K. Lee, Ann. Rep. NMR Spectrosc. 52 (2004) 2. [5] F.M. Marassi, S.J. Opella, J. Magn. Reson. 144 (2000) 150. [6] J. Wang, J. Denny, C. Tian, S. Kim, Y. Mo, F. Kovacs, Z. Song, K. Nishimura, Z. Gan, R. Fu, J.R. Quine, T.A. Cross, J. Magn. Reson. 144 (2000) 162. [7] S.V. Dvinskikh, H. Zimmermann, A. Maliniak, D. Sandstrom, J. Chem. Phys. 122 (2005). [8] Y.F. Wei, D.K. Lee, K.J. Hallock, A. Ramamoorthy, Chem. Phys. Lett. 351 (2002) 42. [9] R.Q. Fu, C.L. Tian, H. Kim, S.A. Smith, T.A. Cross, J. Magn. Reson. 159 (2002) 167. [10] A.A. Nevzorov, S.J. Opella, J. Magn. Reson. 164 (2003) 182. [11] D.K. Lee, T. Narasimhaswamy, A. Ramamoorthy, Chem. Phys. Lett. 399 (2004) 359. [12] K. Nishimura, A. Naito, Chem. Phys. Lett. 402 (2005) 245. [13] A.E. Bennet, C.M. Reinstra, M. Auger, K.V. Lakshmi, R.G. Griffin, J. Chem. Phys. 103 (1995) 6951. [14] M. Lee, W.I. Goldburg, Phys. Rev. A 140 (1965) 1261. [15] M. Mehring, J.S. Waugh, Phys. Rev. B 5 (1972) 3459. [16] A. Bielecki, A.C. Kolbert, H.J.M. de Groot, R.G. Griffin, M.H. Levitt, Adv. Magn. Reson. 14 (1990) 111. [17] M. Ravikumar, A. Ramamoorthy, Chem. Phys. Lett. 286 (1998) 199. [18] M. Bak, J.T. Rasmussen, N.C. Nielsen, J. Magn. Reson. 147 (2000) 296.
Broadband-PISEMA solid-state NMR spectroscopy K. Yamamoto, D.K. Lee, A. Ramamoorthy
*
Biophysics Research Division and Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA Received 22 February 2005; in final form 18 March 2005 Available online 13 April 2005
Abstract The measurement of heteronuclear dipolar couplings using a 2D separated-local-field technique, Polarization Inversion Spin Exchange at the Magic Angle (PISEMA), is a unique way to determine the structure, dynamics and topology of molecules in solid-state. However, the resolution and sensitivity of PISEMA are highly dependent on the offset frequency of protons. To overcome this difficulty, in this study, a broadband-PISEMA pulse sequence is proposed. Experimental data from a single crystal and simulated results suggest that the new sequence compensates the offset effects. This is accomplished using a pair of 180° pulses that invert the spin-locked magnetization of I and S nuclei after certain number of SEMA cycles in the t1 period. In addition, unlike PISEMA, BB-PISEMA provides offset-independent dipolar coupling line shapes even when low rf fields are applied. Ó 2005 Elsevier B.V. All rights reserved.
1. Introduction Polarization Inversion Spin Exchange at the Magic Angle (PISEMA) is a 2D separated-local-field (SLF) experiment that correlates the heteronuclear dipolar coupling with the chemical shift of low c nuclei [1–4]. The unique advantage of this method over other types of SLF experiments is that it has a large scaling factor (0.816; that is 18.4% of the heteronuclear dipolar coupling is suppressed by the pulse sequence in the t1 period) and provides very narrow dipolar coupling spectral lines. Therefore, this technique is commonly used to determine the backbone conformation and topology of aligned molecules such as membrane- associated peptides and proteins [4–6]. Recent studies have extended the application of this technique to studies under magic angle spinning (MAS) [2,4] and also to characterize liquid crystalline materials [7]. The process SEMA has also been utilized in the development of new solid-state NMR methods [4,8]. However, the major disadvantage of PISEMA is its dependency on 1H offset [4]. Ramped *
Corresponding author. Fax: +1 734 763 2307. E-mail address: [email protected] (A. Ramamoorthy).
0009-2614/$ - see front matter Ó 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2005.03.082
spin-lock of low c nuclei during SEMA [9] and a new pulse sequence, SAMMY [10], are shown to be relatively less sensitive to offset effects of protons. Another way to overcome offset effects is to use very high power rf pulses. However, use of high rf power is technically demanding and it is not desirable for biological samples as they are sensitive to heat. These problems are severe for solid-state NMR studies at high magnetic fields. Recent studies have demonstrated the use of time averaged nutation (TAN) concept to reduce the power requirement in PISEMA applications [11,12]. In this study, we demonstrate a modified broadband-PISEMA (BBPISEMA) sequence to compensate the offset and other pulse imperfection effects.
2. Results and discussion Spin-locking 1H magnetization at the magic angle in the SEMA part (i.e., the t1 period) of the PISEMA pulse sequence (Fig. 1a) results in the magnetization exchange via the heteronuclear dipolar coupling that is scaled by sinhm (where hm is the magic angle). On the other hand, the effective field direction of the flip-flop Lee–Goldburg
290
K. Yamamoto et al. / Chemical Physics Letters 407 (2005) 289–293
Fig. 1. Pulse sequences for spin exchange at magic angle: (a) the original PISEMA sequence; (b) a broadband SEMA (or BB-SEMA) sequence that can be used in the t1 period of the PISEMA sequence to compensate the offset effects. The p pulses in the sequence can be separated by several cycles of FFLG (flip-flop Lee–Goldburg) depending on the required spectral width in the dipolar coupling dimension.
(FFLG) sequence depends on the offset frequency, and therefore, the degree of 1H–1H dipolar decoupling, the efficiency of the 1H spin-lock, the scaling factor, and the rate of spin exchange under the SEMA sequence depend on the 1H offset value as revealed by the previous studies [4,9]. For example, 1H offset increases the extent of spin-locked magnetization that do not participate in the spin exchange process during SEMA which reduces the signal intensity by increasing the zero frequency peak intensity in the dipolar coupling spectrum. It also increases the scaling factor and the line width [4,10]. The dephasing of the 1H magnetization from the magic angle direction due to offset effects during the FFLG sequence of SEMA can be eliminated by simultaneously inverting the direction of the spin-locked magnetization of both I and S nuclei as shown in Fig. 1b. Therefore, it is desirable to have two p pulses for each dwell time in the t1 dimension of the PISEMA experiment as shown in Fig. 1. Since a p pulse is also ap-
plied in the S spin channel, the BB-SEMA sequence should also be less sensitive to S spin offset and effects due to rf field inhomogeneity. All experiments were performed on a Chemagnetics/ Varian Infinity 400 MHz spectrometer using a 5 mm triple-resonance MAS probe at room temperature. An rf field strength of 50 kHz at 35.355 kHz offset was used to set-up the Lee–Goldburg condition in the 1H channel with an effective field strength of 61.2 kHz. An rf field strength of 61.2 kHz was used to spin-lock the 15N magnetization during SEMA and 75 kHz was used to decouple protons using the TPPM sequence [13] during 15N signal acquisition. Since there are two magnetically non-equivalent sites in an unit cell of NAVL (n-acetyl15 15 15 L- N-valyl-L- N-leucine) crystal, the N chemical shift spectrum (data not shown) consists of four well resolved lines from two different amide–15N sites of the dipeptide. 2D experiments were performed for various offset values of 1H and 15N nuclei in order to compare the efficiency
K. Yamamoto et al. / Chemical Physics Letters 407 (2005) 289–293
291
Fig. 2. 2D correlation of 15N chemical shift and 1H–15N dipolar coupling of n-acetyl-L-15N-valyl-L-15N-leucine single crystal at an arbitrary orientation with respective to the external magnetic field: (a) PISEMA and (b) BB-PISEMA spectra. The 2D PISEMA spectrum: (a) was obtained from 128 t1 increments with a dwell time of 32.66 ls (4.18 ms acquisition) while the BB-PISEMA spectrum; (b) was the resultant of 48 t1 increments with a dwell time of 40.86 ls (1.961 ms acquisition). Other experimental parameters include a 500 ls contact time for ramp-CP, four scans and a recycle delay of 3 s.
of the regular PISEMA and the BB-PISEMA pulse sequences. Sample 2D spectra for an arbitrary orientation of the crystal are given in Fig. 2. (a) Experimental
(b) Simulations Dipolar Coupling ( kHz)
18
Experimentally measured (Fig. 3a) and simulated (Fig. 3b) dipolar coupling values as a function of 1H offset in PISEMA and BB-PISEMA sequences are given in
14
PISEMA 25
1H-15N
10
35
BB-PISEMA
15 6
-30
0
-30
-40
-20
0
20
40
Percentage of 1H Offset during t 1
Scaling Factor of BB-SEMA
(c) Simulations 0.8
0.7
0.6
0
10
20
π pulse power/power of LG
Fig. 3. (a) Experimentally measured and (b) Simulated 1H–15N dipolar coupling values as a function of 1H offset. The experimental values were measured from 2D PISEMA (open circles and open rectangles) and BB-PISEMA (filled circles and filled rectangles) spectra of NAVL single crystal obtained under the same experimental conditions. The solid line and dashed lines in (a) belong to two different 15N chemical shift resonances at 83.7 and 102.2 ppm of the spectrum (Fig. 2), respectively. The solid line and the dashed–dotted lines in (b) were simulated for 100 and 75 kHz rf field strengths. (c) Variation of the scaling factor with the ratio of the rf power used for the p and LG pulses in the BB-PISEMA. In the simulations, 100 kHz rf field was used for LG and the p pulse power was varied.
292
K. Yamamoto et al. / Chemical Physics Letters 407 (2005) 289–293
Fig. 3. The power of the p pulse used in the experiment and the simulation was the same as the spin-lock rf power in the respective rf channels. On-resonance scaling factors were 0.816 for PISEMA and 0.73 for BB-PISEMA. The percentage of 1H offset in Fig. 3 is defined as (d/LG)*100; where d is the deliberately set offset of the 1H rf carrier and LG is the offset frequency needed to set the effective field direction of SEMA at the magic angle. For example, 10% offset in Fig. 3 is equal to 3.5355 kHz when LG = 35.355 kHz and mrf = 50 kHz were used. The dipolar couplings measured from the 2D PISEMA spectrum are highly dependent on the 1H offset value (shown in open circles and open rectangles). Even a 10% offset increases the dipolar splitting by 20% of the on-resonance dipolar splitting in the PISEMA spectrum (Fig. 3a). This is because the scaling factor of the FFLG and hence the SEMA sequence change with the 1H offset value [4,14–17]. In fact, at large 1H offset values, the dipolar coupling was not measurable as there was no observable signal in the PISEMA spectrum. On the other hand, as shown in Fig. 3, the introduction of a p pulse (Fig. 1b) suppresses the offset effects (shown in filled circles and filled rectangles). For example, 30% offset in the BB-PISEMA sequence changes the dipolar splitting by 5% of the on-resonance dipolar splitting (Fig. 3a). The offset effects on PISEMA and BB-PISEMA were simulated using SIMPSON [18]. A 1H and 15N spin pair with a dipolar coupling constant of 10 kHz was considered in the simulations. The simulated spectra (not shown) for increasing 1H offset values showed an increase in the intensity of the zero-frequency peak while the intensity of the lines separated by the dipolar coupling value decreased. This is because the increase in the offset field increases the magnetization spinlocked along an axis that is away from the magic angle direction. Since this spin-locked magnetization does not participate in the spin exchange process, it results in a strong zero-frequency peak in the x1 dimension of the 2D PISEMA spectrum. As seen from Fig. 3b, simulated results are in good agreement with the experimental data even though the homonuclear dipolar couplings among protons were not included in the simulations. Two different rf field strengths (75 and 100 kHz) were used in the simulations to test the rf power dependency of these pulse sequences. Results suggest that the efficiency of the BB-PISEMA is independent of the rf power used for as much as 35% offset. On the other hand, the efficiency of the PISEMA is better when a higher rf power was used and becomes poor as the rf power was reduced. The scaling factor of the BB-PISEMA depends on the rf power of the p pulse and also on the ratio of the duration of FFLG and p pulse width. As shown in Fig. 3c, it approaches the scaling factor of the PISEMA sequence (0.8165) as the power of the p pulse is increased. Inter-
estingly, the scaling factor of BB-PISEMA is 0.73 even for a moderate p pulse length of 8.2 ls. Sample dipolar coupling slices for two different 1H offset values are given in Fig. 4. The line width for onresonance PISEMA appears to be better than that of BB-PISEMA as longer t1 acquisition was used for PISEMA experiments; whereas experiments with similar t1 acquisition resulted in comparable line widths. However, as shown in Fig. 5, the shape and resolution of PISEMA spectral lines highly depend on the 1H offset value. The offset effect is even stronger when the 1 H–15N dipolar splitting is smaller. For example, the dipolar coupling slices corresponding to other two sites (at 137.2 and 167.8 ppm) that have smaller dipolar coupling values (Fig. 2) are in the noise level even for an offset of ±2 kHz. On the other hand, as shown in Fig. 4, the dipolar coupling spectra measured using the BB-PISEMA sequence are insensitive to the offset effects. Experiments were also carried out to examine the role of 15N offset and the results are given in Fig. 5. PISEMA is less sensitive to 15N offset as compared to 1H offset at least when strong rf fields are used. Our experimental results suggest that the BB-PISEMA sequence (shown in filled circles and filled squares in Fig. 5) is more tolerant to 15N offset effects than the regular PISEMA sequence (shown in empty circles and empty squares in Fig. 5). PISEMA data are not shown for large 15N offset values as the dipolar couplings were not observable in the PISEMA spectra. These results demonstrate that the new sequence, BB-PISEMA, compensates the offset effects of both the dipolar coupled nuclei. This is
Fig. 4. 1H–15N dipolar coupling spectra obtained at different 1H offset values from PISEMA (A) and BB-PISEMA (B). Sites A and B correspond to a 15N chemical shift value of 83.7 and 102.2 ppm, respectively.
K. Yamamoto et al. / Chemical Physics Letters 407 (2005) 289–293
12
of the sequence can be improved by reducing the p pulse width. We believe that the BB-PISEMA sequence will be highly valuable in the structural studies of biological molecules such as membrane-associated peptides and proteins and also liquid crystalline materials.
10
Acknowledgement
Dipolar Coupling ( kHz)
14
1H- 15N
293
This research was supported by the research funds from NIH (AI054515 to A.R).
8
References 6
-20
-10 15N
0
10
20
Offset (kHz) during t 1
Fig. 5. Variation of experimentally measured 1H–15N dipolar coupling values as a function of 15N offset in PISEMA (dashed lines) and BBPISEMA (solid lines) sequences. Open (PISEMA) and filled (BBPISEMA) circles correspond to the Site A at 83.7 ppm, and open (PISEMA) and filled (BB-PISEMA) squares correspond to the Site B at 102.2 ppm.
significant for experiments under low rf power conditions and/or at high magnetic fields.
3. Conclusion We have demonstrated that the newly designed pulse sequence, BB-PISEMA, is capable of compensating offset effects of both the heteronuclei. The dipolar splitting and the line shape of BB-PISEMA spectra do not depend on offset values for a bandwidth of 10 kHz when a 50 kHz rf field was used and this bandwidth increases with an increase in the rf power. The scaling factor of this sequence is identical to PISEMA when an ideal pulse is used while it is
[1] C.H. Wu, A. Ramamoorthy, S.J. Opella, J. Magn. Reson. A 109 (1994) 270. [2] A. Ramamoorthy, S.J. Opella, Solid State NMR Spectrosc. 4 (1995) 387. [3] A. Ramamoorthy, C.H. Wu, S.J. Opella, J. Magn. Reson. 140 (1999) 131. [4] A. Ramamoorthy, Y. Wei, D.K. Lee, Ann. Rep. NMR Spectrosc. 52 (2004) 2. [5] F.M. Marassi, S.J. Opella, J. Magn. Reson. 144 (2000) 150. [6] J. Wang, J. Denny, C. Tian, S. Kim, Y. Mo, F. Kovacs, Z. Song, K. Nishimura, Z. Gan, R. Fu, J.R. Quine, T.A. Cross, J. Magn. Reson. 144 (2000) 162. [7] S.V. Dvinskikh, H. Zimmermann, A. Maliniak, D. Sandstrom, J. Chem. Phys. 122 (2005). [8] Y.F. Wei, D.K. Lee, K.J. Hallock, A. Ramamoorthy, Chem. Phys. Lett. 351 (2002) 42. [9] R.Q. Fu, C.L. Tian, H. Kim, S.A. Smith, T.A. Cross, J. Magn. Reson. 159 (2002) 167. [10] A.A. Nevzorov, S.J. Opella, J. Magn. Reson. 164 (2003) 182. [11] D.K. Lee, T. Narasimhaswamy, A. Ramamoorthy, Chem. Phys. Lett. 399 (2004) 359. [12] K. Nishimura, A. Naito, Chem. Phys. Lett. 402 (2005) 245. [13] A.E. Bennet, C.M. Reinstra, M. Auger, K.V. Lakshmi, R.G. Griffin, J. Chem. Phys. 103 (1995) 6951. [14] M. Lee, W.I. Goldburg, Phys. Rev. A 140 (1965) 1261. [15] M. Mehring, J.S. Waugh, Phys. Rev. B 5 (1972) 3459. [16] A. Bielecki, A.C. Kolbert, H.J.M. de Groot, R.G. Griffin, M.H. Levitt, Adv. Magn. Reson. 14 (1990) 111. [17] M. Ravikumar, A. Ramamoorthy, Chem. Phys. Lett. 286 (1998) 199. [18] M. Bak, J.T. Rasmussen, N.C. Nielsen, J. Magn. Reson. 147 (2000) 296.