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Toward complete 1H NMR spectra in proteins
JOURNAL
OF MAGNETIC
77, f 66-169
RESONANCE
(1988)
COMMUNICATIONS Toward Complete ‘H NMR Spectra in Proteins STEPHEN
C. BROWN, PAUL L. WEBER, AND LUCIANO MUELLER
Smith Kline & French Laboratories, Research and Development Division, Mail code: L940, P.O. Box 1539, King of Prussia, Pennsylvania 19406-0939 Received September
14, 1987
The elucidation of protein structures by NMR depends upon the ability to record two-dimensional ‘H spectra in Hz0 solution (I), which requires suppression of the large Hz0 resonance. Numerous suppression methods have been proposed (2-25) recently, and one might question the necessity of yet another solvent suppression technique. Nevertheless, we feel that the sequences outlined in this paper offer to produce the most complete two-dimensional proton spectra in biopolymers yet obtained. There are three general strategies to overcome the problem of dynamic range in Hz0 solution, by selectively exciting (2-15) the resonances of interest, by saturating the solvent peak (16-21), or by selective broadening of the water resonance (22, 23). These techniques work well as long as no resonances of interest overlap with the water line. This is not the case in proteins where the solvent peak occurs in the range of C”H resonances, being a well-known problem hindering complete resonance assignments in proteins. For example, in ubiquitin five C”H resonances are obliterated by water saturation at 30°C. The missing peaks are typically located by recording another complete data set at a second temperature. This, however, is both tedious and timeconsuming, and it is not always possible due to sample stability. A few techniques (24-26) have been introduced recently that eliminate the water peak by combining a homogeneity spoiling pulse in the mixing period and semiselective “jump and return” pulses at the beginning of the detection period. This scheme fully recovers all NH-C*H cross peaks but leaves a large region around the water resonance bleached out, and the homospoiled water signal tends to introduce noise. Furthermore, these spectra usually suffer from a large baseline roll due to the requirement of large linear phase correction in Fz. Some attempts (12, 13) to eliminate the phase roll of selectively excited FIDs were only moderately successful in high sensitivity probes due to excessive feedback between the solvent signal and the probe circuitry (i.e., radiation damping). However, the refocusing technique introduced by Bax and co-workers appears to be the most promising one in this respect, since it minimizes the excitation of solvent signal during the echo pulse (26). Selective saturation (16-21) on the other hand provides good suppression of the water signal combined with uniform excitation across the entire spectrum. Unfortu0022-2364/88
$3.00
Copyright 0 1988 by Academic F’res~,Inc. All rights of reproduction in any form reserved.
166
167
COMMUNICATIONS
nately a band of cross peaks centered around the water resonance along F2 is obliterated. Otting and Wiithrich (27) recently showed that double-quantum spectra provide complete fingerprints even in the presence of water irradiation, since double-quantum coherences are derived from transverse magnetization of at least two participating spins. This principle of double-quantum excitation, however, cannot be easily extended to other two-dimensional techniques such as the NOESY experiment. We demonstrate here that all bleached C”H resonances can be recovered by one of the simple pulse sequences as depicted in Fig. 1. These sequences can be easily inserted into the preparation period of any two-dimensional NMR experiment. There is one caveat: these sequences only work in systems where T2 4 T, , and where T, is dominated by dipolar relaxation, because tlhe proton magnetizations in the protein decay with T, during tP (see Fig. 1). In this scheme the irradiating field is switched off for a period tP before the start of the two-dimensional experiment, allowing the spin system to approach an internal equilibrium. Equilibration occurs during this period between the saturated C”H resonances and their surrounding neighbors through dipolar cross relaxation. The composite population inversion pulse in the middle of t, of sequence A prevents the relaxation recovery of the solvent peak. Since the spins of interest also relax, t, must be kept much shorter than the longitudinal relaxation time T1. For short 1, the loss in sensitivity due to relaxation is proportional to M/MO = 1 - tp/T, . Sequence B in Fig. 1 reduces this sensitivity loss to a certain extent since the spin system relaxes toward the equilibrium in the second half of the preparation period. We tested this method by obtaining DQF-COSY and NOESY spectra of ubiquitin (8 mA4 dissolved in 25 mM acetate buffer in 90% HZ0 at 30°C) using sequence A in Fig. 1. In the NOESY pulse sequence there was no solvent irradiation during the mixing time, during which water relaxation was prevented by a composite 180” pulse in the center oft,. Figure 2a depicts the fingerprint region of a superposition of the DQF-COSY and the NOESY spectrum. The area which would have been obscured lays between the two broken lines parallel to F2. This fingerprint region now contains all expected NH-C”H cross peaks including V5, L15, V17, D2 1, and R54 (28) which previously could not be observed at this temperature.
I
90i180i90x
-1.2
set+ +---t,/2-++---t,/2+I
I I ,
I -c----4
FIG. 1. Pulse sequences used to recover saturated resonances under the solvent peak. Both sequences can be inserted in the preparation period of any two-dimensional experiment. Note that the vertical broken line marks the end of SCUBA and the beginning of the two-dimensional experiment.
168
COMMUNICATIONS
I
,
I
9.5
I
I
9.0
8.5
8.0
7.5
7.0
5.5
5.0
4.5
4.0
3.5
I
8.5
I
ppm
b
ppm
FIG. 2. Superposition of the DQF-COSY and NOESY spectrum of ubiquitin in 90% Hz0 at 30°C. Sequence A was used in these experiments. The mixing time in the NOESY experiment was 150 ms; the preparation t, was 60 ms. 400 t, FIDs were collected in each experiment with 32 scans pert, value. (a) Fingerprint region, the area between the horizontal broken lines contains the recovered cross peaks. (b) Region near the water peak, many C”H-C”H cross peaks can be observed in Hz0 due to better suppression. All spectra were collected on a Jeol GX500 spectrometer operating at 500.1 MHz proton frequency. Data processing was done on a pVAX II using D. Hare’s sofiware (29). F2 ridges were removed by a polynomial baseline correction algorithm.
169
COMMUNICATIONS
The proposed solvent suppression technique works best when it is combined with phase locking of the saturation RF as previously described by Hoult (16) and by Zuiderweg et al. (21). This not on.ly eliminates many artifacts induced by the residual water signal but also narrows the range along F1 obliterated by the solvent resonance in the C”H region as illustrated in Fig. 2b. We have shown that the proplosed peak recovery method works well for proteins as small as ubiquitin (76 amino acid residues). As pointed out above, our sequence can be incorporated in any two-dimensional NMR experiment. This sequence can be referred to as SCUBA (stimulated cross peaks under bleached alphas) (30), since it allows protons to breathe under water. ACKNOWLEDGMENTS We thank Drs. S. Crook, D. Ecker, and T. Butt and Mr. J. Marsh for providing the ubiquitin This research has been supported in part by NIH Program Project Grant GM39526.
sample.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
K. WOTHRICH, “NMR of Proteins and Nucleic Acids,” Wiley, New York, 1986. A. G. REDFIELD, S. D. KUNZ, AND E. K. RALPH, J. Magn. Reson. 19, 114 (1975). J. DADOK AND R. F. SPRECHER, J. Magn. Reson. 13,243 (1974). P. PLATEAU AND M. G&RON, J. Am. Chem. Sot. 104,731O (1982). V. SKLENAR AND Z. STARCUK, J. Magn. Reson. 50,495 (1982). P. PLATEAU, C. DUMAS, AND M. GU~RON, J. Magn. Reson. 54,46 (1983). D. L. TURNER, J. Magn. Reson. 54, 146 (1983). P. J. HORE, J. Mugn. Resort. 54, 539 (1983). P. J. HORE, J. Mugn. Reson. 55, 283 (1983). P. J. HORE, J. Magn. Reson. 56, 535 (1984). M. MCCOY AND W. S. WARREN, Chem. Phys. Lett. 133, 165 (1987). M. H. LEVITT AND M. F. ROBERTS, J. Magn. Reson. 71,576 (1987). G. J. GALLOWAY, L. J. HASELER, M. F. MARSHMAN, D. H. WILLIAMS, AND D. M. DODDRELL, Magn. Reson. 74, 184 (1987). M. P. HALL AND P. J. HORE, J. Magn. Reson. 70, 350 (1986). C. WANG AND A. PARDI, J. Magn. .Reson. 71, 154 (1987). D. HOULT, J. Magn. Reson. 21, 337 (1976). A. KUMAR, G. WAGNER, R. R. ERNST, AND K. WOTHRICH, Biochem. Biophys. Rex Commun.
J.
96,
1156 (1980). 18. M. 19. V. 20. G. 21. E. 22. R. 23. D. 24. A. 25. E. 26. V. 27. G. 28.(a)
OHUCHI, M. HOSONO, K. MATUSHITA, AND M. IMANARI, J. Magn. Reson. 43,499 (1981). J. BASUS, J. Magn. Reson. 60, 138 (1984). WIDER, R. V. HOSUR, AND K. WUTHRICH, J. Magn. Reson. 52, 130 (1983). R. P. ZUIDERWEG, K. HALLENGA, AND E. T. OLEJNICZAK, J. Magn. Reson. 70, 336 (1986). G. BRYANT AND T. M. EADS, J. Magn. Reson. 64,312 (1985). L. RABENSTEIN AND S. FAN, Anal. Chem. 58, 3178 (1986). L. SCHWARTZ AND J. D. CUTNELL, J. Magn. Reson. 53, 398 (1983). R. P. ZUIDERWEG, J. Magn. Reson. 71, 283 (1987). SKLENAR, B. R. BROOKS, G. ZON, AND A. BAX, FEBS Lett. 216,249 (1987). OTTING AND K. WOTHRICH, J. Magn. Reson. 66, 359 (1986). D. DI STEPHANO AND A. J. WAND, Biochemistry 26,70 (1987); (b) P. L. WEBER, S. C. BROWN, AND L. MUELLER, Biochemistry 26, 72 (1987).
29. FINMR
program licensed by Hare Research, Inc., 14810 216th Ave. N. E., Woodinville, Washington
98072. 30. L. MUELLER,
R. A. SCHIKSNIS,
AND S. J. OPELLA,
J. Magn.
Reson.
66, 379
(1986).
OF MAGNETIC
77, f 66-169
RESONANCE
(1988)
COMMUNICATIONS Toward Complete ‘H NMR Spectra in Proteins STEPHEN
C. BROWN, PAUL L. WEBER, AND LUCIANO MUELLER
Smith Kline & French Laboratories, Research and Development Division, Mail code: L940, P.O. Box 1539, King of Prussia, Pennsylvania 19406-0939 Received September
14, 1987
The elucidation of protein structures by NMR depends upon the ability to record two-dimensional ‘H spectra in Hz0 solution (I), which requires suppression of the large Hz0 resonance. Numerous suppression methods have been proposed (2-25) recently, and one might question the necessity of yet another solvent suppression technique. Nevertheless, we feel that the sequences outlined in this paper offer to produce the most complete two-dimensional proton spectra in biopolymers yet obtained. There are three general strategies to overcome the problem of dynamic range in Hz0 solution, by selectively exciting (2-15) the resonances of interest, by saturating the solvent peak (16-21), or by selective broadening of the water resonance (22, 23). These techniques work well as long as no resonances of interest overlap with the water line. This is not the case in proteins where the solvent peak occurs in the range of C”H resonances, being a well-known problem hindering complete resonance assignments in proteins. For example, in ubiquitin five C”H resonances are obliterated by water saturation at 30°C. The missing peaks are typically located by recording another complete data set at a second temperature. This, however, is both tedious and timeconsuming, and it is not always possible due to sample stability. A few techniques (24-26) have been introduced recently that eliminate the water peak by combining a homogeneity spoiling pulse in the mixing period and semiselective “jump and return” pulses at the beginning of the detection period. This scheme fully recovers all NH-C*H cross peaks but leaves a large region around the water resonance bleached out, and the homospoiled water signal tends to introduce noise. Furthermore, these spectra usually suffer from a large baseline roll due to the requirement of large linear phase correction in Fz. Some attempts (12, 13) to eliminate the phase roll of selectively excited FIDs were only moderately successful in high sensitivity probes due to excessive feedback between the solvent signal and the probe circuitry (i.e., radiation damping). However, the refocusing technique introduced by Bax and co-workers appears to be the most promising one in this respect, since it minimizes the excitation of solvent signal during the echo pulse (26). Selective saturation (16-21) on the other hand provides good suppression of the water signal combined with uniform excitation across the entire spectrum. Unfortu0022-2364/88
$3.00
Copyright 0 1988 by Academic F’res~,Inc. All rights of reproduction in any form reserved.
166
167
COMMUNICATIONS
nately a band of cross peaks centered around the water resonance along F2 is obliterated. Otting and Wiithrich (27) recently showed that double-quantum spectra provide complete fingerprints even in the presence of water irradiation, since double-quantum coherences are derived from transverse magnetization of at least two participating spins. This principle of double-quantum excitation, however, cannot be easily extended to other two-dimensional techniques such as the NOESY experiment. We demonstrate here that all bleached C”H resonances can be recovered by one of the simple pulse sequences as depicted in Fig. 1. These sequences can be easily inserted into the preparation period of any two-dimensional NMR experiment. There is one caveat: these sequences only work in systems where T2 4 T, , and where T, is dominated by dipolar relaxation, because tlhe proton magnetizations in the protein decay with T, during tP (see Fig. 1). In this scheme the irradiating field is switched off for a period tP before the start of the two-dimensional experiment, allowing the spin system to approach an internal equilibrium. Equilibration occurs during this period between the saturated C”H resonances and their surrounding neighbors through dipolar cross relaxation. The composite population inversion pulse in the middle of t, of sequence A prevents the relaxation recovery of the solvent peak. Since the spins of interest also relax, t, must be kept much shorter than the longitudinal relaxation time T1. For short 1, the loss in sensitivity due to relaxation is proportional to M/MO = 1 - tp/T, . Sequence B in Fig. 1 reduces this sensitivity loss to a certain extent since the spin system relaxes toward the equilibrium in the second half of the preparation period. We tested this method by obtaining DQF-COSY and NOESY spectra of ubiquitin (8 mA4 dissolved in 25 mM acetate buffer in 90% HZ0 at 30°C) using sequence A in Fig. 1. In the NOESY pulse sequence there was no solvent irradiation during the mixing time, during which water relaxation was prevented by a composite 180” pulse in the center oft,. Figure 2a depicts the fingerprint region of a superposition of the DQF-COSY and the NOESY spectrum. The area which would have been obscured lays between the two broken lines parallel to F2. This fingerprint region now contains all expected NH-C”H cross peaks including V5, L15, V17, D2 1, and R54 (28) which previously could not be observed at this temperature.
I
90i180i90x
-1.2
set+ +---t,/2-++---t,/2+I
I I ,
I -c----4
FIG. 1. Pulse sequences used to recover saturated resonances under the solvent peak. Both sequences can be inserted in the preparation period of any two-dimensional experiment. Note that the vertical broken line marks the end of SCUBA and the beginning of the two-dimensional experiment.
168
COMMUNICATIONS
I
,
I
9.5
I
I
9.0
8.5
8.0
7.5
7.0
5.5
5.0
4.5
4.0
3.5
I
8.5
I
ppm
b
ppm
FIG. 2. Superposition of the DQF-COSY and NOESY spectrum of ubiquitin in 90% Hz0 at 30°C. Sequence A was used in these experiments. The mixing time in the NOESY experiment was 150 ms; the preparation t, was 60 ms. 400 t, FIDs were collected in each experiment with 32 scans pert, value. (a) Fingerprint region, the area between the horizontal broken lines contains the recovered cross peaks. (b) Region near the water peak, many C”H-C”H cross peaks can be observed in Hz0 due to better suppression. All spectra were collected on a Jeol GX500 spectrometer operating at 500.1 MHz proton frequency. Data processing was done on a pVAX II using D. Hare’s sofiware (29). F2 ridges were removed by a polynomial baseline correction algorithm.
169
COMMUNICATIONS
The proposed solvent suppression technique works best when it is combined with phase locking of the saturation RF as previously described by Hoult (16) and by Zuiderweg et al. (21). This not on.ly eliminates many artifacts induced by the residual water signal but also narrows the range along F1 obliterated by the solvent resonance in the C”H region as illustrated in Fig. 2b. We have shown that the proplosed peak recovery method works well for proteins as small as ubiquitin (76 amino acid residues). As pointed out above, our sequence can be incorporated in any two-dimensional NMR experiment. This sequence can be referred to as SCUBA (stimulated cross peaks under bleached alphas) (30), since it allows protons to breathe under water. ACKNOWLEDGMENTS We thank Drs. S. Crook, D. Ecker, and T. Butt and Mr. J. Marsh for providing the ubiquitin This research has been supported in part by NIH Program Project Grant GM39526.
sample.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
K. WOTHRICH, “NMR of Proteins and Nucleic Acids,” Wiley, New York, 1986. A. G. REDFIELD, S. D. KUNZ, AND E. K. RALPH, J. Magn. Reson. 19, 114 (1975). J. DADOK AND R. F. SPRECHER, J. Magn. Reson. 13,243 (1974). P. PLATEAU AND M. G&RON, J. Am. Chem. Sot. 104,731O (1982). V. SKLENAR AND Z. STARCUK, J. Magn. Reson. 50,495 (1982). P. PLATEAU, C. DUMAS, AND M. GU~RON, J. Magn. Reson. 54,46 (1983). D. L. TURNER, J. Magn. Reson. 54, 146 (1983). P. J. HORE, J. Mugn. Resort. 54, 539 (1983). P. J. HORE, J. Mugn. Reson. 55, 283 (1983). P. J. HORE, J. Magn. Reson. 56, 535 (1984). M. MCCOY AND W. S. WARREN, Chem. Phys. Lett. 133, 165 (1987). M. H. LEVITT AND M. F. ROBERTS, J. Magn. Reson. 71,576 (1987). G. J. GALLOWAY, L. J. HASELER, M. F. MARSHMAN, D. H. WILLIAMS, AND D. M. DODDRELL, Magn. Reson. 74, 184 (1987). M. P. HALL AND P. J. HORE, J. Magn. Reson. 70, 350 (1986). C. WANG AND A. PARDI, J. Magn. .Reson. 71, 154 (1987). D. HOULT, J. Magn. Reson. 21, 337 (1976). A. KUMAR, G. WAGNER, R. R. ERNST, AND K. WOTHRICH, Biochem. Biophys. Rex Commun.
J.
96,
1156 (1980). 18. M. 19. V. 20. G. 21. E. 22. R. 23. D. 24. A. 25. E. 26. V. 27. G. 28.(a)
OHUCHI, M. HOSONO, K. MATUSHITA, AND M. IMANARI, J. Magn. Reson. 43,499 (1981). J. BASUS, J. Magn. Reson. 60, 138 (1984). WIDER, R. V. HOSUR, AND K. WUTHRICH, J. Magn. Reson. 52, 130 (1983). R. P. ZUIDERWEG, K. HALLENGA, AND E. T. OLEJNICZAK, J. Magn. Reson. 70, 336 (1986). G. BRYANT AND T. M. EADS, J. Magn. Reson. 64,312 (1985). L. RABENSTEIN AND S. FAN, Anal. Chem. 58, 3178 (1986). L. SCHWARTZ AND J. D. CUTNELL, J. Magn. Reson. 53, 398 (1983). R. P. ZUIDERWEG, J. Magn. Reson. 71, 283 (1987). SKLENAR, B. R. BROOKS, G. ZON, AND A. BAX, FEBS Lett. 216,249 (1987). OTTING AND K. WOTHRICH, J. Magn. Reson. 66, 359 (1986). D. DI STEPHANO AND A. J. WAND, Biochemistry 26,70 (1987); (b) P. L. WEBER, S. C. BROWN, AND L. MUELLER, Biochemistry 26, 72 (1987).
29. FINMR
program licensed by Hare Research, Inc., 14810 216th Ave. N. E., Woodinville, Washington
98072. 30. L. MUELLER,
R. A. SCHIKSNIS,
AND S. J. OPELLA,
J. Magn.
Reson.
66, 379
(1986).