- Email: [email protected]
HMQC-NOESY-HMQC, a three-dimensional NMR experiment which allows detection of nuclear overhauser effects between protons with overlapping signals
JOlJRNAL
OF M4GNETIC
RESONANCE
90,420-425
( 1990)
HMQC-NOESY-HMQC, a Three-Dimensional NMR Experiment Which Allows Detection of Nuclear Overhauser Effects between Protons with Overlapping Signals T.
FRENKIEL,*
C.
BAUER,*
M.
D.
Received
CARR,?
June
B. BIRDSALL,?
AND
J. FEENEY
t
27. 1990
In recent years the structures of many small proteins have been determined by twodimensional NMR, using methods pioneered by Wiithrich et al. (I ). These methods depend exclusively on data from proton NMR experiments and despite their success it has become clear that they are more difficult to apply and less likely to work for proteins with molecular weights of more than about 15,000. The deleterious effects of greater molecular size arise both from the large number of signals and from the increase in the linr~tidths of the signals: among other things these effects result in spectra which have a much higher degree of overlap. This is particularly acute in proteins with a high helical content, in which, for example, the majority of the amide protons are expected to resonate within a range of just one part per million. Several different strategies for overcoming this problem have been proposed. Among the most promising are those that depend upon nonspecific near-complete isotopic substitution of 15N for 14N or 13C for “C, in conjunction with two-dimensional NMR experiments which exploit the shift range of the heteronuclei to separate the proton signals (2-8). An alternative and potentially very powerful strategy is the use of threedimensional NMR to alleviate spectral overlap (9-12). The combination of threedimensional NMR with heteronuclear labeling is proving particularly effective (1318), and an important example ofthis approach is the 3D NOESY-HMQC experiment, developed by Marion et ~11.(14) and Zuiderweg and Fesik (15) for use with “Nlabeled proteins. This experiment may be thought of as generating a 15N-edited NOESY spectrum, with signals spread out in a third dimension according to the lSN shift of the directly coupled amide nitrogens. More formally, a proton H, which has an NOE to an amide proton Hi, will generate a cross peak in the NOESY-HMQC experiment centered at frequency coordinates (h,, nb, hi,), where h, and hb are the chemical shifts of H, and Hh, respectively, and nb is the chemical shift of the “N nucleus that is directly coupled to Hi,. It is clear that by spreading the signals according to the nitrogen shifts this experiment would allow the Ha-HI, interaction to be identified even if there were one or more additional protons which had the same shift as Hi,. However, if H, and Hi, themselves overlap the NOESY-HMQC experiment does not allow an NOE interaction between them to be detected: in this case the presence of the characteristic cross peak at (h,, nb, hb) would be masked by the coincident and more intense “di420
421
COMMUNICATIONS
agonal” peak at (I&, ?$,, hb). In this Communication we describe a complementary 3D experiment (HMQC-NOESY-HMQC) which allows NOES between overlapping amide proton signals to be detected as long as the amide nitrogen shifts are not also degenerate. The key to this experiment is that both members of each pair of crossrelaxing amide protons are characterized by their nitrogen shifts. Cross-peak formation in the HMQC-NOESY-HMQC experiment can be understood by reference to the pulse sequence shown in Fig. 1. Initially, an HMQC sequence ( 19, 20) is used to generate amide proton magnetization modulated by the frequency of its own nitrogen as a function oft, . This is followed by a simple proton 90” pulse to regenerate proton z magnetization together with a fixed mixing time 7 during which cross-relaxation can transfer the modulation to nearby protons. If any of these are also amide protons a second HMQC sequence then provides indirect detection of their nitrogen frequencies (as a function of t2) with direct detection of the proton frequencies themselves during t3. In terms of the notation developed above the cross peak representing an NOE from H, to Hb will appear at frequency coordinates (n,, nb, hb). The reciprocal nature of the cross-relaxation process ensures that there will be a corresponding cross peak at (nb, n,, h,) due to transfer from Hb to H,. The 3D spectrum will also contain peaks which are equivalent to diagonal peaks, generated by magnetization which remains associated with the same nucleus throughout the mixing time: an amide proton such as H, will give a “diagonal” peak of this type at (n,, n,, h,). It follows that, as long as n, and nb are distinct, overlap of h, and hb does not interfere with detection of the Ha--HI, interaction: NOES between overlapping or nonoverlapping protons can be detected with equal ease. Conversely, if n, and nb do overlap detection of the H,-Hb interaction is prevented by the diagonal peak, irrespective of the shift difference between h, and hb: for this reason the experiment must be regarded as complementary to the previous methods. As in all three-dimensional experiments phase-cycling options are limited because of the large number of increments which need to be carried out. Table 1 gives details of a basic eight-step cycle in which the first four steps select signals that have arisen
90,
180+x
90x
180+x
go@,
N-15
goa*
90,
FIG. 1. Pulse sequence for the NOESY-HMQC-NOESY irradiation which was applied during the NOESY mixing been omitted from the diagram.
9003
90x
experiment. In the interest of clarity solvent time T and during the delay between scans has
422
COMMUNICATIONS TABLE Basic Scan
Phase-Cycle
I
for the NOESY-HMQC-NOESY 42
Experiment $3
Receiver
via NH double- or zero-quantum coherence, while the first and second set of four scans together eliminate artifacts which could arise from single-quantum proton magnetization present during the NOESY mixing time. It is beneficial to increase the cycle length by a factor of four to accommodate independent alternation of the phases of the two proton 180” pulses. To obtain absorption-mode lineshapes with sign discrimination in F, and Fz we employ the TPPI method of Marion and Wtithrich (21): phases & and & are independently incremented in 90” steps with each successive increment in t, and t2, respectively. The HMQC-NOESY-HMQC experiment has been used to investigate the amide protons of “N-labeled Lactobaci//us cash dihydrofolate reductase (DHFR) in a 1: 1 complex with methotrexate ( MTX ). This protein has a molecular weight of 18,300 and was isolated from Exhrrichia co/i into which the gene for L. casei DHFR had been cloned, following procedures described previously (22, 23). In order to achieve a high level of “N labeling the cells were grown on a minimal medium containing ammonium sulfate enriched to 99% in 15N as the sole nitrogen source. NMR experiments were carried out on a 5 mA4 sample of the complex in 90% H20-10% DZO with 500 mA4 potassium chloride and 50 mM potassium phosphate at pH 6.4. Data were acquired on a Bruker AM-500 spectrometer fitted with a BSV-7 transmitter, a BFX-5 X-nucleus decoupler, and a 5 mm inverse-detection probe. Water suppression was provided by irradiation with DANTE sequences (24) during the recycle delay and during the NOESY mixing time. Good baselines were obtained by a combination of(i) setting the receiver phase to give sine modulation in t3 (25. 2h), (ii) gating the receiver on within a few microseconds of the final radiofrequency pulse, and (iii) adjusting the delay between the last pulse and the start of data acquisition to give the flattest baseline as adjudged from 1D spectra obtained with the 3D sequence. This procedure eliminates the need for software baseline corrections. During the acquisition period a GARP decoupling sequence (27) was applied to the 15N spins with a radiofrequency field strength of 1.7 kHz: this provided excellent decoupling and required only 1.9 W of radiofrequency power. Standard Bruker acquisition software was used to increment t2 in conjunction with a PASCAL program to increment 1,. Data were transferred by Ethernet to a Silicon Graphics IRIS 3 120 workstation prior to processing and plotting using software written in-house. Some illustrative plots are shown in Fig. 2. These F,-F? cross sections are taken
423
COMMUNICATIONS
(a)
F2=121.2
(b)
ppm
F2=111.9
ppm
(cl
F2=105.8
ppm
Thr34,Gln33 \
Gln33,Ala32 \ @-------I
110
t
\
Gln33.ihr34
115
$
120
Val35,Thr34
c Ala32,Gln33 0
1
7.5
125
1
I
I
I
I
I
I
8.0
7.5
7.0
a.5
a.0
7.5
7.0
pm a.5
a.0
7.5
7.0
F3
FIG. 2. Representative F1-F3 cross sections from a 3D NOESY-HMQC-NOESY spectrum of DHFRMTX. For this experiment the HMQC 1 delay was set to 5 ms and the NOESY mixing time to 100 ms. Proton chemical shifts are referenced indirectly to DSS while the “N shift scale is arbitrary. The cross peaks which have been labeled arise from sequential NH-NH NOES covering the residues from Ala 32 to Val 35.
from a much larger data set covering 35 ppm in each of the nitrogen dimensions and 7 ppm in the proton dimension. For the complete data set over 50 NH-NH NOES could be detected. The assignments given in the figure were deduced from this experiment in conjunction with others and will be described in detail in a forthcoming paper. Acquisition of the data took four days using 84 X 84 increments, 32 scans per increment, and 5 12 points per scan. The strongest features on the plots are the rows of peaks which have approximately equal F, and F2 coordinates. These peaks convey the same information as a 2D HMQC experiment and are also analogous to the diagonal peaks of a NOESY spectrum since they arise primarily from magnetization which remains within the same NH group throughout the sequence, as described earlier. The slightly zig-zag appearance of these rows results from the tails of peaks from adjacent cross sections. Part (a) of the figure is the cross section at Fz = 12 1.2 ppm, the chemical shift of the amide nitrogen of Ala 32. The amide proton of this residue resonates at 7.81 ppm and the plot shows that this proton has an NOE from an amide proton whose nitrogen resonates at 111.9 ppm. Inspection of the cross section at this Fz frequency, Fig. 2b, confirms the existence of this NOE and establishes the resonant frequency of the second amide proton (7.89 ppm). The same analysis procedure can be used to identify an NOE between this proton and an amide proton at 7.3 1 ppm with an amide nitrogen shift of 105.8 ppm, as shown in parts (b) and (c) of the figure. Although the amide protons at 7.81 ppm (Ala 32) and 7.89 ppm (Gln 33) are not completely degenerate the relatively small shift difference between them has made it impossible for us to detect this NOE in either 2D NOESY or 3D NOESY-HMQC spectra.
424
COMMUNICATIONS
The general principle underlying the HMQC-NOESY-HMQC experiment is that the signal which represents an interaction between two protons may advantageously be characterized by the resonant frequencies of the heteronuclei to which the protons are directly coupled. In this Communication we have shown how this may be used with 15N as the heteronucleus to detect NOES between amide protons which have overlapping resonances. With 13C-labeled proteins the same method could be useful for detecting NOES between aliphatic protons since these signals can also be subject to severe overlap. Mixed experiments involving one “C dimension and one “N dimension can also be envisaged: these could be of value in studies of interactions between amide protons and aliphatic protons in larger proteins. Scalar interactions between protons could be investigated with a version of the experiment in which the NOESY mixing period is replaced by a period of isotropic mixing, as in the twodimensional TOCSY and HOHAHA experiments (28, 29). Finally, an additional proton evolution period could be introduced to make a four-dimensional experiment in which both proton frequencies and both heteronuclear frequencies would be measured. This would allow a proton-proton interaction to be detected if the signals of either the protons or the heteronuclei were overlapping. ACKNOWLEDGMENTS We acquired
are grateful and
to G. Ostler
processed
using
and
J. McCormick
the facilities
for expert
of the MRC
technical
Biomedical
NMR
assistance.
The
NMR
data
were
Centre.
REFERENCES 1. K. WOTHRICH, “NMR of Proteins 2. A. BAX AND M. A. WEISS, J. Mqn. S. 4. 5. 6.
and
11. H. OSCHKINAT, 1.2. G. 13. S. 14. D.
16
E. D.
I7 18.
M. E.
19. L. 20.
Acids.”
Wiley.
New
York,
1986.
H. SENN, G. OTTING, 1090 ( 1987). M. RANCE, P. E. WRIGH’I, B. A. MESSERLE, AND L. D. FIELD. J. Am. C’hem. Sot 109, 1591 (1987). S. W. FESIK, R. T. GAMPE, AND T. W. RO~KWAY, J. Magn. Rcson. 74, 366 ( 1987). L. P. MCINTOSH, F. W. DAHLQ~JIS’I, AND A. G. REDFIELD, J B;ww~ Swuc~. fI!x 5, 21 ( 1987).
7. D. A. TORCHIA, S. W. SPARKS. AND 8. D. A. TORCHIA, S. W. SPARKS, P. E. Y. H. D. PLANT, T. H. MARECI, M. D. April 13- 17, 1986.” 10. G. W. VUISTER AND R. BOELENS, J.
IS.
Nucleic
Re,son. 71, 571 (1987). AND K. WOTHRICH, J Am C‘hrm. Sot. 109,
A.
A. BAX, Biochemi.stq~ 28, 5509 ( 1989). YOIJNG, AND A. BAX, J. Am C‘hcm. SW. COCKMAI\;, AND W. S. BREY, “27th ENC,
Magn.
11 I, 8315 Baltimore,
( 1989). Maryland.
Rcw~t7. 73, 328 ( 1987).
C. GRIESINGER. P. J. KRAULIS, 0. W. SORENSEN, R. R. ERNST, A. M. GRONENBORN, AND G. M. CLORE. Nature jlondon) 322, 374 ( 1988). W. VUISTER, R. BOELENS, AND R. KAPTEIN, J. Map Rcwn. 80, 176 (1988). W. FESIK AND E. R. P. ZUIDERWEG, J. Map. Rrson. 78, 588 (1988). MARION, L. E. KAY, S. W. SPARKS, D. A. TORCHIA, AND A. BAX, J .4rn. Che!n. SOCK. 111, 1515 (1989). R. P. ZUIDERWEG AND S. W. FESIK, Biochemistg~ 28, 2387 ( 1989). MARION, P. C. DRISCOLI., L. E. KAY, P. T. WINGFIELD, A. BAX, A. M. GRONENBORN, AND G. M. CLORE, Biochemistr~~ 28, 6 150 ( 1989). IKURA. L. E. KAY, R. TSCHUDIN, AND A. BAX. J. Magn. Rc.wn. 86, 204 ( 1990). R. P. Z~JIDERWEG, L. P. MCINTOSH, F. W. DAHLQUIST. AND S. W. FESIK, J Mann Reton. 86,210 (1990). MOL.L.ER. J. .Am. C’hrm SOC. 101, 4481 ( 1979). BAX, R. H. GRIFFEY, AND B. I.. HAWKINS, J ,Ma,qn. Recon. 55, 301 ( 1983).
COMMUNICATIONS
425
23
D. MARION AND K. WOTHRICH, Biochem. Bioph.w Rex Commun. 113, 967 (1983). J. G. DANN, G. OSTLER, R. A. BJUR, R. W. KING, P. SCUDDER, P. C. TURNER. G. C. K. ROBERTS. A. S. V. BURGEN, AND N. G. L. HARDING, Biochem. J. 157, 559 ( 1976). J. ANDREWS. G. M. CLORE, R. W. DAVIES. A. M. GRONENBORN, B. GRONENBORN, D. KALDERON.
24. 25. 26
P. C. PAPADOPOULOS, S. SCHAFER, P. F. G. SIMS, AND R. STANCOMBE. Genr35,217 ( 1985). G. A. MORRIS AND R. FREEMAN, J. Mugti. Re.son. 29, 433 ( 1978). G. OTTING, H. WIDMER, G. WAGNER, AND K. WOTHRICH, J. Mugn Reson. 66, 187 (1986). D. MARION AND A. BAX, J. Mugn. Reson 79, 352 (1988).
21. 22.
27. A. J. SHAKA, P. B. BARKER, AND R. FREEMAN, J. Magn. Re.von. 64, 547 (1985). 2X L. BRAUNS~HWEILER AND R. R. ERNST, J. Alup. Rcwn. 53, 521 (1983). 2Y. A. BAX AND D. G. DAVIES, J. Map. Rewn 65, 355 ( 1985).
OF M4GNETIC
RESONANCE
90,420-425
( 1990)
HMQC-NOESY-HMQC, a Three-Dimensional NMR Experiment Which Allows Detection of Nuclear Overhauser Effects between Protons with Overlapping Signals T.
FRENKIEL,*
C.
BAUER,*
M.
D.
Received
CARR,?
June
B. BIRDSALL,?
AND
J. FEENEY
t
27. 1990
In recent years the structures of many small proteins have been determined by twodimensional NMR, using methods pioneered by Wiithrich et al. (I ). These methods depend exclusively on data from proton NMR experiments and despite their success it has become clear that they are more difficult to apply and less likely to work for proteins with molecular weights of more than about 15,000. The deleterious effects of greater molecular size arise both from the large number of signals and from the increase in the linr~tidths of the signals: among other things these effects result in spectra which have a much higher degree of overlap. This is particularly acute in proteins with a high helical content, in which, for example, the majority of the amide protons are expected to resonate within a range of just one part per million. Several different strategies for overcoming this problem have been proposed. Among the most promising are those that depend upon nonspecific near-complete isotopic substitution of 15N for 14N or 13C for “C, in conjunction with two-dimensional NMR experiments which exploit the shift range of the heteronuclei to separate the proton signals (2-8). An alternative and potentially very powerful strategy is the use of threedimensional NMR to alleviate spectral overlap (9-12). The combination of threedimensional NMR with heteronuclear labeling is proving particularly effective (1318), and an important example ofthis approach is the 3D NOESY-HMQC experiment, developed by Marion et ~11.(14) and Zuiderweg and Fesik (15) for use with “Nlabeled proteins. This experiment may be thought of as generating a 15N-edited NOESY spectrum, with signals spread out in a third dimension according to the lSN shift of the directly coupled amide nitrogens. More formally, a proton H, which has an NOE to an amide proton Hi, will generate a cross peak in the NOESY-HMQC experiment centered at frequency coordinates (h,, nb, hi,), where h, and hb are the chemical shifts of H, and Hh, respectively, and nb is the chemical shift of the “N nucleus that is directly coupled to Hi,. It is clear that by spreading the signals according to the nitrogen shifts this experiment would allow the Ha-HI, interaction to be identified even if there were one or more additional protons which had the same shift as Hi,. However, if H, and Hi, themselves overlap the NOESY-HMQC experiment does not allow an NOE interaction between them to be detected: in this case the presence of the characteristic cross peak at (h,, nb, hb) would be masked by the coincident and more intense “di420
421
COMMUNICATIONS
agonal” peak at (I&, ?$,, hb). In this Communication we describe a complementary 3D experiment (HMQC-NOESY-HMQC) which allows NOES between overlapping amide proton signals to be detected as long as the amide nitrogen shifts are not also degenerate. The key to this experiment is that both members of each pair of crossrelaxing amide protons are characterized by their nitrogen shifts. Cross-peak formation in the HMQC-NOESY-HMQC experiment can be understood by reference to the pulse sequence shown in Fig. 1. Initially, an HMQC sequence ( 19, 20) is used to generate amide proton magnetization modulated by the frequency of its own nitrogen as a function oft, . This is followed by a simple proton 90” pulse to regenerate proton z magnetization together with a fixed mixing time 7 during which cross-relaxation can transfer the modulation to nearby protons. If any of these are also amide protons a second HMQC sequence then provides indirect detection of their nitrogen frequencies (as a function of t2) with direct detection of the proton frequencies themselves during t3. In terms of the notation developed above the cross peak representing an NOE from H, to Hb will appear at frequency coordinates (n,, nb, hb). The reciprocal nature of the cross-relaxation process ensures that there will be a corresponding cross peak at (nb, n,, h,) due to transfer from Hb to H,. The 3D spectrum will also contain peaks which are equivalent to diagonal peaks, generated by magnetization which remains associated with the same nucleus throughout the mixing time: an amide proton such as H, will give a “diagonal” peak of this type at (n,, n,, h,). It follows that, as long as n, and nb are distinct, overlap of h, and hb does not interfere with detection of the Ha--HI, interaction: NOES between overlapping or nonoverlapping protons can be detected with equal ease. Conversely, if n, and nb do overlap detection of the H,-Hb interaction is prevented by the diagonal peak, irrespective of the shift difference between h, and hb: for this reason the experiment must be regarded as complementary to the previous methods. As in all three-dimensional experiments phase-cycling options are limited because of the large number of increments which need to be carried out. Table 1 gives details of a basic eight-step cycle in which the first four steps select signals that have arisen
90,
180+x
90x
180+x
go@,
N-15
goa*
90,
FIG. 1. Pulse sequence for the NOESY-HMQC-NOESY irradiation which was applied during the NOESY mixing been omitted from the diagram.
9003
90x
experiment. In the interest of clarity solvent time T and during the delay between scans has
422
COMMUNICATIONS TABLE Basic Scan
Phase-Cycle
I
for the NOESY-HMQC-NOESY 42
Experiment $3
Receiver
via NH double- or zero-quantum coherence, while the first and second set of four scans together eliminate artifacts which could arise from single-quantum proton magnetization present during the NOESY mixing time. It is beneficial to increase the cycle length by a factor of four to accommodate independent alternation of the phases of the two proton 180” pulses. To obtain absorption-mode lineshapes with sign discrimination in F, and Fz we employ the TPPI method of Marion and Wtithrich (21): phases & and & are independently incremented in 90” steps with each successive increment in t, and t2, respectively. The HMQC-NOESY-HMQC experiment has been used to investigate the amide protons of “N-labeled Lactobaci//us cash dihydrofolate reductase (DHFR) in a 1: 1 complex with methotrexate ( MTX ). This protein has a molecular weight of 18,300 and was isolated from Exhrrichia co/i into which the gene for L. casei DHFR had been cloned, following procedures described previously (22, 23). In order to achieve a high level of “N labeling the cells were grown on a minimal medium containing ammonium sulfate enriched to 99% in 15N as the sole nitrogen source. NMR experiments were carried out on a 5 mA4 sample of the complex in 90% H20-10% DZO with 500 mA4 potassium chloride and 50 mM potassium phosphate at pH 6.4. Data were acquired on a Bruker AM-500 spectrometer fitted with a BSV-7 transmitter, a BFX-5 X-nucleus decoupler, and a 5 mm inverse-detection probe. Water suppression was provided by irradiation with DANTE sequences (24) during the recycle delay and during the NOESY mixing time. Good baselines were obtained by a combination of(i) setting the receiver phase to give sine modulation in t3 (25. 2h), (ii) gating the receiver on within a few microseconds of the final radiofrequency pulse, and (iii) adjusting the delay between the last pulse and the start of data acquisition to give the flattest baseline as adjudged from 1D spectra obtained with the 3D sequence. This procedure eliminates the need for software baseline corrections. During the acquisition period a GARP decoupling sequence (27) was applied to the 15N spins with a radiofrequency field strength of 1.7 kHz: this provided excellent decoupling and required only 1.9 W of radiofrequency power. Standard Bruker acquisition software was used to increment t2 in conjunction with a PASCAL program to increment 1,. Data were transferred by Ethernet to a Silicon Graphics IRIS 3 120 workstation prior to processing and plotting using software written in-house. Some illustrative plots are shown in Fig. 2. These F,-F? cross sections are taken
423
COMMUNICATIONS
(a)
F2=121.2
(b)
ppm
F2=111.9
ppm
(cl
F2=105.8
ppm
Thr34,Gln33 \
Gln33,Ala32 \ @-------I
110
t
\
Gln33.ihr34
115
$
120
Val35,Thr34
c Ala32,Gln33 0
1
7.5
125
1
I
I
I
I
I
I
8.0
7.5
7.0
a.5
a.0
7.5
7.0
pm a.5
a.0
7.5
7.0
F3
FIG. 2. Representative F1-F3 cross sections from a 3D NOESY-HMQC-NOESY spectrum of DHFRMTX. For this experiment the HMQC 1 delay was set to 5 ms and the NOESY mixing time to 100 ms. Proton chemical shifts are referenced indirectly to DSS while the “N shift scale is arbitrary. The cross peaks which have been labeled arise from sequential NH-NH NOES covering the residues from Ala 32 to Val 35.
from a much larger data set covering 35 ppm in each of the nitrogen dimensions and 7 ppm in the proton dimension. For the complete data set over 50 NH-NH NOES could be detected. The assignments given in the figure were deduced from this experiment in conjunction with others and will be described in detail in a forthcoming paper. Acquisition of the data took four days using 84 X 84 increments, 32 scans per increment, and 5 12 points per scan. The strongest features on the plots are the rows of peaks which have approximately equal F, and F2 coordinates. These peaks convey the same information as a 2D HMQC experiment and are also analogous to the diagonal peaks of a NOESY spectrum since they arise primarily from magnetization which remains within the same NH group throughout the sequence, as described earlier. The slightly zig-zag appearance of these rows results from the tails of peaks from adjacent cross sections. Part (a) of the figure is the cross section at Fz = 12 1.2 ppm, the chemical shift of the amide nitrogen of Ala 32. The amide proton of this residue resonates at 7.81 ppm and the plot shows that this proton has an NOE from an amide proton whose nitrogen resonates at 111.9 ppm. Inspection of the cross section at this Fz frequency, Fig. 2b, confirms the existence of this NOE and establishes the resonant frequency of the second amide proton (7.89 ppm). The same analysis procedure can be used to identify an NOE between this proton and an amide proton at 7.3 1 ppm with an amide nitrogen shift of 105.8 ppm, as shown in parts (b) and (c) of the figure. Although the amide protons at 7.81 ppm (Ala 32) and 7.89 ppm (Gln 33) are not completely degenerate the relatively small shift difference between them has made it impossible for us to detect this NOE in either 2D NOESY or 3D NOESY-HMQC spectra.
424
COMMUNICATIONS
The general principle underlying the HMQC-NOESY-HMQC experiment is that the signal which represents an interaction between two protons may advantageously be characterized by the resonant frequencies of the heteronuclei to which the protons are directly coupled. In this Communication we have shown how this may be used with 15N as the heteronucleus to detect NOES between amide protons which have overlapping resonances. With 13C-labeled proteins the same method could be useful for detecting NOES between aliphatic protons since these signals can also be subject to severe overlap. Mixed experiments involving one “C dimension and one “N dimension can also be envisaged: these could be of value in studies of interactions between amide protons and aliphatic protons in larger proteins. Scalar interactions between protons could be investigated with a version of the experiment in which the NOESY mixing period is replaced by a period of isotropic mixing, as in the twodimensional TOCSY and HOHAHA experiments (28, 29). Finally, an additional proton evolution period could be introduced to make a four-dimensional experiment in which both proton frequencies and both heteronuclear frequencies would be measured. This would allow a proton-proton interaction to be detected if the signals of either the protons or the heteronuclei were overlapping. ACKNOWLEDGMENTS We acquired
are grateful and
to G. Ostler
processed
using
and
J. McCormick
the facilities
for expert
of the MRC
technical
Biomedical
NMR
assistance.
The
NMR
data
were
Centre.
REFERENCES 1. K. WOTHRICH, “NMR of Proteins 2. A. BAX AND M. A. WEISS, J. Mqn. S. 4. 5. 6.
and
11. H. OSCHKINAT, 1.2. G. 13. S. 14. D.
16
E. D.
I7 18.
M. E.
19. L. 20.
Acids.”
Wiley.
New
York,
1986.
H. SENN, G. OTTING, 1090 ( 1987). M. RANCE, P. E. WRIGH’I, B. A. MESSERLE, AND L. D. FIELD. J. Am. C’hem. Sot 109, 1591 (1987). S. W. FESIK, R. T. GAMPE, AND T. W. RO~KWAY, J. Magn. Rcson. 74, 366 ( 1987). L. P. MCINTOSH, F. W. DAHLQ~JIS’I, AND A. G. REDFIELD, J B;ww~ Swuc~. fI!x 5, 21 ( 1987).
7. D. A. TORCHIA, S. W. SPARKS. AND 8. D. A. TORCHIA, S. W. SPARKS, P. E. Y. H. D. PLANT, T. H. MARECI, M. D. April 13- 17, 1986.” 10. G. W. VUISTER AND R. BOELENS, J.
IS.
Nucleic
Re,son. 71, 571 (1987). AND K. WOTHRICH, J Am C‘hrm. Sot. 109,
A.
A. BAX, Biochemi.stq~ 28, 5509 ( 1989). YOIJNG, AND A. BAX, J. Am C‘hcm. SW. COCKMAI\;, AND W. S. BREY, “27th ENC,
Magn.
11 I, 8315 Baltimore,
( 1989). Maryland.
Rcw~t7. 73, 328 ( 1987).
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