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The Application of Selective-Excitation Pulse Sequences in NMR Spectroscopy of Paramagnetic Proteins
JOURNAL OF MAGNETIC RESONANCE, ARTICLE NO.
Series B 110, 202–204 (1996)
0032
COMMUNICATIONS The Application of Selective-Excitation Pulse Sequences in NMR Spectroscopy of Paramagnetic Proteins MARIO PICCIOLI Department of Chemistry, University of Florence, via G. Capponi, 7, 50121 Florence, Italy Received June 26, 1995; revised December 4, 1995
Paci and co-workers (1) have recently reported that selective-excitation pulse sequences (2–4), which are extensively used in the case of diamagnetic systems, can improve the detection of cross peaks in the case of far-shifted, fastrelaxing signals in paramagnetic metalloproteins. However, this view is inconsistent with current ‘‘state-of-the-art’’ NMR methodology and instrumentation for the study of paramagnetic proteins. To briefly demonstrate this, I will consider the same example used by the authors of the above-mentioned article. Dicopper, dicobalt, superoxide dismutase (Cu2Co2SOD) is a paramagnetic, dimeric protein, about 32 kDa, which is extremely suitable for NMR investigation (5). The NMR spectrum shows broad, fast-relaxing signals that are shifted far downfield (up to 70 ppm) with respect to the diamagnetic envelope. In general, the value of T 2 dramatically influences the detection of these signals, which may be barely observable with respect to the noise, and so optimization of S/N ratio is important when observing broad and relaxed resonances. The rationale behind the use of selective-excitation pulse sequences is that the S/N ratio of the hyperfine-shifted signals is limited by the presence of the very intense signals of the diamagnetic part of the protein. The S/N ratio in a single-transient acquisition can be improved by increasing the receiver gain of the spectrometer so long as the preamplifier noise stays below the threshold noise of the analog-to-digital converter (A/D). If the dynamic range of the digitizer is not fully used, the noise originated by the digitizer itself will be added to the FID at a relatively high level, resulting in a poor dynamic range. If two largely unequal noise levels are added, the resulting noise will be only marginally higher than the larger of the two, while the summation of two equivalent noise levels results in a 3 dB increase. By increase of the receiver gain, the S/N ratio of the spectrum will improve until the receiver gain is such that the preamplifier noise matches or surpasses the A/D’s noise level. From this point on, a further increase 1064-1866/96 $12.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
m4982$7341
01-31-96 22:45:57
in gain does not change the S/N ratio of the spectrum, since both the noise and signal are equally enhanced and digitized. Since digitizers are preceded by audio filters, which usually reduce the receiver bandwidth to the spectral region of interest, the desirable minimum receiver gain changes only slightly with the spectral width. If, for example, the gain was set at 256 for a spectral width of 20 ppm, it could be reduced to 128 for a spectral width of 80 ppm, since the noise level follows the square root of the bandwidth. Figure 1 shows the S/N ratio obtained for a broad far-shifted signal in the spectrum of Cu2Co2SOD as a function of the receiver gain used. The data were collected at 600 MHz on a Bruker AMX spectrometer, using a 16 bit AD converter on an approximately 0.5 mM Cu2Co2SOD sample in 90% H2O, 10% D2O. The carrier was positioned at the water frequency, and a spectral window of 125 kHz (dwell time of 4 ms) was used. Spectra were acquired with 128 scans, with presaturation of the water signal and a fast repetition rate (approximately 10 acquisitions per second). There is almost no improvement on increasing the receiver gain from 64 to 128, and definitely no improvement from 128 to 256. No dependence of the S/N ratio on the excitation profile was observed with offset frequencies in the range 0– 40 kHz. Analogous results are obtained if the experiment is performed with alternative pulse sequences. The data reported in Fig. 1 have been reproduced with the superWEFT pulse sequence (6), which alter the relative intensities of signals on the basis of their relaxation rates. When 20 transients per second are recorded, the receiver gain can be increased to 1024 without any observable signal distortion. An identical S/N ratio for hyperfine-shifted resonances is observed for receiver gain values ranging from 128 to 1024. The experiment was also repeated using an offresonance DANTE sequence for solvent suppression (7, 8). With this sequence, carrier position can be placed far from the solvent signal and a smaller spectral window can be used. When the carrier was placed at about 31 ppm and a spectral window of 54 kHz (dwell time 9.2S ms) was used, similar data to those reported in Fig. 1 were obtained.
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203
FIG. 1. Part of the 1D spectrum (downfield region) of Cu2Co2SOD as a function of the spectrometer receiver gain. The spectrum was acquired with 128 transients on a Bruker AMX 600 spectrometer. Experimental details are reported in the text. The broad signal at about 67 ppm has a T 1 value of about 1.5 ms and linewidth (at 600 MHz) of approximately 1500 Hz. Receiver gain values used are indicated in the figure.
These results indicate that, even for dilute proteins in H2O solutions, a simple solvent presaturation or WEFT-type pulse sequence (6, 9) allows one to reach an optimal spectrometer receiver gain, beyond which the S/N ratio no longer improves. Therefore the recently reported application of selective-excitation pulse sequences to suppress diamagnetic signals do not provide any practical advantage in terms of either the S/N or the experimental information that can be obtained.
m4982$7341
01-31-96 22:45:57
maga
If the reading pulse of a NOESY experiment is replaced by a binomial type pulse (10), where the delays are adjusted to suppress the entire spectral region 10–0 ppm, there is an ‘‘a priori’’ loss of information which arises from the fact that the excitation profile will filter out half of the cross peaks of the two-dimensional spectrum and will significantly influence the relative intensity of the other half. This prevents the quantitative use of NOESY information. Admittedly, the
AP: Mag Res
204
COMMUNICATIONS
reduction of intense signals in the diamagnetic region is expected to decrease T 1 noise in that region. However, this effect is irrelevant because the reduction of T 1 noise occurs in a spectral region that does not contain connectivities between paramagnetic and diamagnetic signals. Furthermore, selective pulse sequences do not address one of the central issues in the investigation of paramagnetic molecules: the identification of signals that are affected by paramagnetic relaxation but are not shifted far from the diamagnetic region (11). Pulse sequences that are based on the differential relaxation properties of signals may be used to enhance, among signals in the diamagnetic region, those which are influenced to some extent by the hyperfine interaction (9, 12). This is a crucial advantage with respect to sequences based on frequency-selective filters. Finally, one may observe that efforts to improve the sensitivity of experiments involving hyperfine-shifted, fast-relaxing signals should primarily focus on the tuning of all experimental parameters with respect to the connectivities of interest (13–16). Mixing times as well as t2max and t1max should be chosen according to the T 1 and T 2 values of the signals of interest. Indeed, if relaxation times are known, it is always possible to optimize the experimental conditions to detect a cross peak or a set of cross peaks with similar relaxation behavior. Because of the fast decay of paramagnetic signals, their S/N ratio can also be varied by selecting the number of acquired data points (in both dimensions) that should be apodized and Fourier transformed. It can be easily demonstrated that a missetting of any of these parameters can have a dramatic effect on the detection of cross peaks involving fast-relaxing signals.
m4982$7341
01-31-96 22:45:57
maga
ACKNOWLEDGMENTS The continuous advice and support of Professor Ivano Bertini is gratefully acknowledged. I am grateful to Claudio Luchinat, David L. Turner, Roberta Pierattelli, and Antonio Donaire for the many discussions. I also thank Professor James A. Cowan for his careful reading of this manuscript.
REFERENCES 1. M. Sette, A. Desideri, G. Rotilio, and M. Paci, J. Magn. Reson. B 105, 91 (1994). 2. P. Plateau and M. Gueron, J. Am. Chem. Soc. 104, 7310 (1982). 3. M. Von Kienlin, M. Decorps, J. P. Albrand, M. F. Foray, and P. Blondet, J. Magn. Reson. 76, 169 (1988). 4. V. Sklena´rˇ and A. Bax, J. Magn. Reson. 74, 469 (1987). 5. I. Bertini, C. Luchinat, and M. Piccioli, Prog. NMR Spectrosc. 26, 91 (1994). 6. T. Inubushi and E. D. Becker, J. Magn. Reson. 51, 128 (1983). 7. G. A. Morris and R. Freeman, J. Magn. Reson. 29, 433 (1978). 8. L. E. Kay and A. Bax, J. Magn. Reson. 86, 110 (1990). 9. Z. G. Chen, J. S. de Ropp, G. Hernandez, and G. N. La Mar, J. Am. Chem. Soc. 116, 8772 (1994). 10. D. L. Turner, J. Magn. Reson. 54, 146 (1983). 11. I. Bertini, P. Turano, and A. J. Vila, Chem. Rev. 93, 2833 (1993). 12. I. Bertini, A. Dikiy, C. Luchinat, M. Piccioli, and D. Tarchi, J. Magn. Reson. B 103, 278 (1994). 13. L. Banci, I. Bertini, and C. Luchinat, in ‘‘Methods in Enzymology’’ (T. L. James and N. J. Oppenheimer, Eds.) p. 485, Academic Press, London, 1994. 14. C. Luchinat and M. Piccioli, in ‘‘NMR of Paramagnetic Macromolecules. NATO ASI Series’’ (G. N. La Mar, Ed.), p. 1, Kluwer Academic, Dordrecht, 1995. 15. G. N. La Mar and J. S. de Ropp, in ‘‘Biological Magnetic Resonance’’ (L. J. Berliner and J. Reuben, Eds.), Vol. 12, p. 1, Plenum Press, New York, 1993. 16. A. V. Xavier, D. L. Turner, and H. Santos, Methods Enzymol. 227, 1 (1993).
AP: Mag Res
Series B 110, 202–204 (1996)
0032
COMMUNICATIONS The Application of Selective-Excitation Pulse Sequences in NMR Spectroscopy of Paramagnetic Proteins MARIO PICCIOLI Department of Chemistry, University of Florence, via G. Capponi, 7, 50121 Florence, Italy Received June 26, 1995; revised December 4, 1995
Paci and co-workers (1) have recently reported that selective-excitation pulse sequences (2–4), which are extensively used in the case of diamagnetic systems, can improve the detection of cross peaks in the case of far-shifted, fastrelaxing signals in paramagnetic metalloproteins. However, this view is inconsistent with current ‘‘state-of-the-art’’ NMR methodology and instrumentation for the study of paramagnetic proteins. To briefly demonstrate this, I will consider the same example used by the authors of the above-mentioned article. Dicopper, dicobalt, superoxide dismutase (Cu2Co2SOD) is a paramagnetic, dimeric protein, about 32 kDa, which is extremely suitable for NMR investigation (5). The NMR spectrum shows broad, fast-relaxing signals that are shifted far downfield (up to 70 ppm) with respect to the diamagnetic envelope. In general, the value of T 2 dramatically influences the detection of these signals, which may be barely observable with respect to the noise, and so optimization of S/N ratio is important when observing broad and relaxed resonances. The rationale behind the use of selective-excitation pulse sequences is that the S/N ratio of the hyperfine-shifted signals is limited by the presence of the very intense signals of the diamagnetic part of the protein. The S/N ratio in a single-transient acquisition can be improved by increasing the receiver gain of the spectrometer so long as the preamplifier noise stays below the threshold noise of the analog-to-digital converter (A/D). If the dynamic range of the digitizer is not fully used, the noise originated by the digitizer itself will be added to the FID at a relatively high level, resulting in a poor dynamic range. If two largely unequal noise levels are added, the resulting noise will be only marginally higher than the larger of the two, while the summation of two equivalent noise levels results in a 3 dB increase. By increase of the receiver gain, the S/N ratio of the spectrum will improve until the receiver gain is such that the preamplifier noise matches or surpasses the A/D’s noise level. From this point on, a further increase 1064-1866/96 $12.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
m4982$7341
01-31-96 22:45:57
in gain does not change the S/N ratio of the spectrum, since both the noise and signal are equally enhanced and digitized. Since digitizers are preceded by audio filters, which usually reduce the receiver bandwidth to the spectral region of interest, the desirable minimum receiver gain changes only slightly with the spectral width. If, for example, the gain was set at 256 for a spectral width of 20 ppm, it could be reduced to 128 for a spectral width of 80 ppm, since the noise level follows the square root of the bandwidth. Figure 1 shows the S/N ratio obtained for a broad far-shifted signal in the spectrum of Cu2Co2SOD as a function of the receiver gain used. The data were collected at 600 MHz on a Bruker AMX spectrometer, using a 16 bit AD converter on an approximately 0.5 mM Cu2Co2SOD sample in 90% H2O, 10% D2O. The carrier was positioned at the water frequency, and a spectral window of 125 kHz (dwell time of 4 ms) was used. Spectra were acquired with 128 scans, with presaturation of the water signal and a fast repetition rate (approximately 10 acquisitions per second). There is almost no improvement on increasing the receiver gain from 64 to 128, and definitely no improvement from 128 to 256. No dependence of the S/N ratio on the excitation profile was observed with offset frequencies in the range 0– 40 kHz. Analogous results are obtained if the experiment is performed with alternative pulse sequences. The data reported in Fig. 1 have been reproduced with the superWEFT pulse sequence (6), which alter the relative intensities of signals on the basis of their relaxation rates. When 20 transients per second are recorded, the receiver gain can be increased to 1024 without any observable signal distortion. An identical S/N ratio for hyperfine-shifted resonances is observed for receiver gain values ranging from 128 to 1024. The experiment was also repeated using an offresonance DANTE sequence for solvent suppression (7, 8). With this sequence, carrier position can be placed far from the solvent signal and a smaller spectral window can be used. When the carrier was placed at about 31 ppm and a spectral window of 54 kHz (dwell time 9.2S ms) was used, similar data to those reported in Fig. 1 were obtained.
202
maga
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203
FIG. 1. Part of the 1D spectrum (downfield region) of Cu2Co2SOD as a function of the spectrometer receiver gain. The spectrum was acquired with 128 transients on a Bruker AMX 600 spectrometer. Experimental details are reported in the text. The broad signal at about 67 ppm has a T 1 value of about 1.5 ms and linewidth (at 600 MHz) of approximately 1500 Hz. Receiver gain values used are indicated in the figure.
These results indicate that, even for dilute proteins in H2O solutions, a simple solvent presaturation or WEFT-type pulse sequence (6, 9) allows one to reach an optimal spectrometer receiver gain, beyond which the S/N ratio no longer improves. Therefore the recently reported application of selective-excitation pulse sequences to suppress diamagnetic signals do not provide any practical advantage in terms of either the S/N or the experimental information that can be obtained.
m4982$7341
01-31-96 22:45:57
maga
If the reading pulse of a NOESY experiment is replaced by a binomial type pulse (10), where the delays are adjusted to suppress the entire spectral region 10–0 ppm, there is an ‘‘a priori’’ loss of information which arises from the fact that the excitation profile will filter out half of the cross peaks of the two-dimensional spectrum and will significantly influence the relative intensity of the other half. This prevents the quantitative use of NOESY information. Admittedly, the
AP: Mag Res
204
COMMUNICATIONS
reduction of intense signals in the diamagnetic region is expected to decrease T 1 noise in that region. However, this effect is irrelevant because the reduction of T 1 noise occurs in a spectral region that does not contain connectivities between paramagnetic and diamagnetic signals. Furthermore, selective pulse sequences do not address one of the central issues in the investigation of paramagnetic molecules: the identification of signals that are affected by paramagnetic relaxation but are not shifted far from the diamagnetic region (11). Pulse sequences that are based on the differential relaxation properties of signals may be used to enhance, among signals in the diamagnetic region, those which are influenced to some extent by the hyperfine interaction (9, 12). This is a crucial advantage with respect to sequences based on frequency-selective filters. Finally, one may observe that efforts to improve the sensitivity of experiments involving hyperfine-shifted, fast-relaxing signals should primarily focus on the tuning of all experimental parameters with respect to the connectivities of interest (13–16). Mixing times as well as t2max and t1max should be chosen according to the T 1 and T 2 values of the signals of interest. Indeed, if relaxation times are known, it is always possible to optimize the experimental conditions to detect a cross peak or a set of cross peaks with similar relaxation behavior. Because of the fast decay of paramagnetic signals, their S/N ratio can also be varied by selecting the number of acquired data points (in both dimensions) that should be apodized and Fourier transformed. It can be easily demonstrated that a missetting of any of these parameters can have a dramatic effect on the detection of cross peaks involving fast-relaxing signals.
m4982$7341
01-31-96 22:45:57
maga
ACKNOWLEDGMENTS The continuous advice and support of Professor Ivano Bertini is gratefully acknowledged. I am grateful to Claudio Luchinat, David L. Turner, Roberta Pierattelli, and Antonio Donaire for the many discussions. I also thank Professor James A. Cowan for his careful reading of this manuscript.
REFERENCES 1. M. Sette, A. Desideri, G. Rotilio, and M. Paci, J. Magn. Reson. B 105, 91 (1994). 2. P. Plateau and M. Gueron, J. Am. Chem. Soc. 104, 7310 (1982). 3. M. Von Kienlin, M. Decorps, J. P. Albrand, M. F. Foray, and P. Blondet, J. Magn. Reson. 76, 169 (1988). 4. V. Sklena´rˇ and A. Bax, J. Magn. Reson. 74, 469 (1987). 5. I. Bertini, C. Luchinat, and M. Piccioli, Prog. NMR Spectrosc. 26, 91 (1994). 6. T. Inubushi and E. D. Becker, J. Magn. Reson. 51, 128 (1983). 7. G. A. Morris and R. Freeman, J. Magn. Reson. 29, 433 (1978). 8. L. E. Kay and A. Bax, J. Magn. Reson. 86, 110 (1990). 9. Z. G. Chen, J. S. de Ropp, G. Hernandez, and G. N. La Mar, J. Am. Chem. Soc. 116, 8772 (1994). 10. D. L. Turner, J. Magn. Reson. 54, 146 (1983). 11. I. Bertini, P. Turano, and A. J. Vila, Chem. Rev. 93, 2833 (1993). 12. I. Bertini, A. Dikiy, C. Luchinat, M. Piccioli, and D. Tarchi, J. Magn. Reson. B 103, 278 (1994). 13. L. Banci, I. Bertini, and C. Luchinat, in ‘‘Methods in Enzymology’’ (T. L. James and N. J. Oppenheimer, Eds.) p. 485, Academic Press, London, 1994. 14. C. Luchinat and M. Piccioli, in ‘‘NMR of Paramagnetic Macromolecules. NATO ASI Series’’ (G. N. La Mar, Ed.), p. 1, Kluwer Academic, Dordrecht, 1995. 15. G. N. La Mar and J. S. de Ropp, in ‘‘Biological Magnetic Resonance’’ (L. J. Berliner and J. Reuben, Eds.), Vol. 12, p. 1, Plenum Press, New York, 1993. 16. A. V. Xavier, D. L. Turner, and H. Santos, Methods Enzymol. 227, 1 (1993).
AP: Mag Res