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Stimulated echo NMR spectra and their use for heteronuclear two-dimensional shift correlation
STIhlULATED ECHO NMR SPECTRA AND THEIR USE FOR HETERONUCLEAR Alfred
G.
22 April 1983
CHEhlfCALPHYSICSLlXl-ERS
Volume96. number 5
TWO-DIMENSIONAL
SHIFT CORRELATION
REDFIELD
Deporrrtrenrs of Physics aladBioclzentisn-y and tlxe Rosemteil Basic Medical Sciences Research Colter. Bmndeis li’al?i!am.hinssachusetrs022.54. US.4 Received 17 January
LGversit_r.
1983
NMR spectra are obtained from Fourier transformation of the latter pti of a Hahn stimulated echo. The scqucnce is 90”-Ta-90”-qy-90”T;i-digitize ~crsus TZ_ This isdemonstrated for transfer RN.4 in Hz0 usirg the scmisclecrbc 3 bt R pulse(90”-7p-900), developed by Plateau and Cueron in place of each 90” pulse. 20 correlation between “N and protons is obtained by applying 3 90°-rt-90” l5 N sequence during q and “N dccouplin,r whifc digitizing. The second Fourier transform is performed with respect to T~ after discarding the imaginary parts of the f=st transform. to obtain a real ZD map. Such a 2D spectrum has been obtained for 5 mM *5NII,CI and several other small molecules The sequence
shouldbe useful for studiesof I5 N labeled macromo!ecules
At the time of his discovery of spitz echoes [I J Hahn also observed and analyzed s~~~?r~~~r~echoes (90”-T,-90”-lb-90°-T,-echO). Some of the recently developed two-dimensional Fourier transform (2D FT) sequences [2] are based on the concept of interference with a spin echo by irradiation on a second species. Here we extend this idea to interference with a stimulated echo_ Incidentally, it is not widely remembered that Hahn’s student, the late Bernard Herzog, performed the first double resonance heteronuclear spin echo experiment in solids years ago [3]. This was essentially a cw precursor to many modern FT techniques. Hahn and co-workers [“s] at the fBM Watson Laboratory at Columbia University also investigated the possible use of stimulated echoes for computer memory applications. A photon echo experiment qualitatively similar to what I will describe was performed by Liao et al. [S]. We developed this method in order to study a 15N labeled macromolecule, transfer RNA (tRNA). Since enviro~ental effects on “N chemical shiftsare small we need as high resolution as possible. Concentration and volume are limited to -2 mM in 0.2 ml (or less, depending on getting enough total sample), so that a proton-observe nitrogen-irradiate experiment was indicated. Griffey et al. 161 have recently labeled 0 009-2614/83/0000-OOOOjS
03.00 0 1983 North-Holland
tRNA with l5 N at the pyrimidine N3 position. and have correlated proton and nitrogen shifts by observing protons while difference-decoupling nitrogen. Resolution of 5 Hz was claimed, but in this experiment the proton lines were well resolved. If the proton lines overlap badly, interpretation will be more difficult_ Thus we sought to develop a 2D FT protonobserve experiment. The technique of Bodenhausen and Ruben 171, essentially an INEPT sequence and an inverse INEPT sequence separated by a nitrogen YO”-T, -90” sequence, should certainly be tried for this type of ap plication. However, it may suffer from some of the following problems: it requires two lS0” pulses on protons and in our probe we expected difficulty in using these clue to xf field inhomogeneity- Esperiments on tRNA must be run in Hz0 which crossrelaxes to the macromolecule protons [S], so that the spins must be allowed to recover for seconds between each sequence. Tile H20 signal would have to be saturated during the sequence to avoid overloading the spectrometer. Or else the sequence would have to be adapted for use with semiselective pulses. Finally, double iNEPT is inherently rather complicated_ The technique we have developed is tentatively called ~AHN~R, in honor of E-L_ Hahn, and the 537
CHEMICAL
Volums 96. number 5
sequence is shou II m fig. I . We first describe how a ~trmui.ued e,3lo ~eq~mx is obtained from Fourier tr~rsforrn (I’T) uf tlirz hitter part of a stimulated e&o ( t
22 April 1983
PHYSICS LETTERS
although there are amplitude variations (fig_ 1 B)- In the version of the J & R sequence that we use for transfer RNA. where the interesting part of the spectrum is far downfield, the two 90” pulse lengths are 40 ps (limited by our low power), -rp is 40 p, the frequency is =c2 kHz downfieid of solvent, and the second 90” pulse is shifted =PO” reiative to the first one. The carrier frequency is thus placed in the center of the interesting region of the spectrum (arrow). This variant of the J & R pulse was used because our proton power is rather low (3 WV). Fig_ 2 shows a single _I 8; R spectrum and a knulated echo spectra obtained in this way. on 200 nM of purified transfer RNA. Unfortunately the amplitude of the echo spectrum is decreased by a factor of about four owing to three reasons: First. a loss of 1f2 because transverse magnetization after the second pulse is lost and does not contribute to the signal. Second, a loss due to rf inhomogeneity &ich is = 1I 1.5 in our spectrometer. Third, a factor expf-2r,ifTz - rb/Tl ) due to relasation. With ra values (2”2 ms) expected to be applicable for 15N work (below) this loss will also be =l /I 5. NMR spectra have not been
j
pulse in tiu,. 1A we used ;f version of the 90”-~~-90~ pulse seq.~ence (called J & R) of Plateau and Gueron
1lo] which produces
no
phase shifts in the spectrum
I
.
I
12
14
p*m
Oownfom
*
I
.
from
\
e
10 OS
Fig_ 2. (A) i?hiR spectrum of ==l mM transfer RNA in 90%
X20, 10% DtO, pf2 6. Sample volume was 0.2 ml and the
spectrum was obtained in 53 min using a single J & R pulse. (B) Stimulated echo spectrum of the same sample using three J & R pulses as in fy. 1A. The spectrum was obtained in about one hour.
Volume96, number S
CHEMICALPHYSICS LE-iTERS
obtained in this way previously to our knowledge, and the echo spectrum may be useful for T2 measurement. Our abiIity to obtain this spectrum also shows, in our opinion, that the J & R pulse can be used to obtain types of 2D spectra in water other than that described below. The first two pulses need not be applied at the same frequency, nor have the same spacing 7 p, as the third. The main disadvantage for 2D NMR will be that since the pulse is semiselective, Tef$OnS of the 2D map will be low in sensitivity. Nowever, in certain applications where storage space or instrument time are Iimited this may be an advantage. For “N spectroscopy the sequence of fig. 1 C is added. The first interval 7a is fared and equal to slightly less than lIZA, where d is the 15NH splitting (~100 Hz). Spectra are obtained and stored for many 71 values. For reasons detailed elsewhere 12, ch. 1 ;I 11, the imaginary parts of incoming spectra obtained as described above are discarded after ET in the first dimension in order to ultimately obtain a real 2D spectrum rather than an absolute value spectrum. Doing so improves the resolution of the 2D spectrum. For each TV, four blocks of data are taken during which the second 90” l5 N pulse is sequenced through four phase shifts, by an integer JZ~times nj2_ A real echo spectrum obtained with r13 = 2 is subtracted from that with jr3 = 0 and stored. and a sirnitar difference spectrum between ?13 = 3 and 1 is stored as imaginary data for the same 71 point. The second FT is then done, with respect to I~ _ It is essential that the ls N be decoupled during digitization. These spectra were obtained on a 270 MHz system described previously Cl?], augmented by an input processor/buffer memory based on an inexpensive SlOO-bus memory [13]. Because of the limited and slow storage available (300 kbyte LINCTAF@, I developed a semitransposed storage method 1141 for the 2D FT. The output map dimensions are limited to 128 Is N by 400 proton points_ The theory of the experiment is straightforward, and will not be given. The idea is that the stimulated echo occurs if there is no change in an individual proton’s precession frequency during rt, and pdsing nitrogen will produce such a change by scrambling the nitrogen orientation for ls N protons. The time -J-, is chosen as usual so that proton isochromats are pointing oppositely for the two “IV orientations just before the second proton pulse, to maximize the effect of Is N perturbation.
“N
22 April 1983
(Coupled I
~50#HZ---4
Fig. 3. (A) Several Iincs of 3 heteronudeat 2I) experimem as shownin Fg. 1C. on SO m&lNH&l in 0.1 M NaOH.The nitrogen carrier frequency was 27 366 880 Hz. the solvent Hz0 proton resonance frequency was 270074 960 i S Hz,
and the times ~a, rP and ~1 were respectively 7 ms, 360 JXS,and O--%6 ms. The proton carrier frequency was 650 Hz downfield from H,O. (B) The PC& spectrum of the set (A) at four times biihcr gain. The noise is entirely due to spectrometer instability and is much Iessfor lines away from the proton peak, as shown in (c) which is also plotted at four times higher gain than (A).
Single lines of 22) maps for 50 mM ls NH4CI in 90% I$ 0, IO% D20 are shown in fig. 3. The unexpected appearance of the ammonium spectrum (missing central line, outer lines equal intensity) can be explained by means of a simple quantum analysis. We have not yet attempted this experiment on tRNA because the required sample has not been prepared, but I believe it will be feasible to obtain useful 2D maps at 270 MHz (5.3 T) with less than 10 mg of tRNA. Most of the noise obvious in fig_ 3B will be decreased in a labeled macromolecule. to be less than thermal noise as in fig. 3C. In principle the method may be useful for natural abundance lSN or 13C spectroscopy, but in practice it may be difficult to avoid noise due to the signal from unlabeled protons, which appears as noise because of spectrometer instability. The factor of two loss in sensitivity inherent in the stimulated echo spectrum mentioned above is theoretically not present in the double INEPT method of Bodenhausen and Ruben [7] _ The lSN resolution may be slightly better because it is the natural resolution for double INEPT but is Iimited by the proton 539
CHEMICAL
Volume 96, number 5
T1 for HAHNDOR. Nitrogen T1 can also be measured with double INEPT and not readily with HAHNDOR. For these reasons doub!e INEPT should stdl be attempted for use with labelled macromolecules despite its complexity and the probable difficultles mentioned earlier. Tills research
\\as supported
by U.S.P.H.S.
grant
GWOlbS. 1 thank Stddhartha Roy for stimulating III mterest in this subject_ Sara Kunz for expert en~~ncc:~n~ 3nd hlmnce Gueron for preliminary infornut1011 on his pulse technique.
PHYSICS
LETTERS
r41 A.G. Anderson, R-L. Garwin. E.L. Hahn. J-W_ Horton, G-L. Tucker and R-hi. WaIker, J_ AppL Phys. 26 (1955) 1324; 27 (1956) 196. ISI P-F. Liao. P. Hu, R. Leigh and S-R. Hartmann, Phys. Rev. A9 (1974)
i 1 ] K.1. II~Iln. Phys Ret. so (1950) 560. 121 A. B.Ix. T\\o JinwxGxxxi nuclcnr magnetic resonxwc xn Iq111ds (Reid& Dordrecht. 1982). [I] 1%Ilerzog_md 11.1. Iiahn. Ph?s Rev. 103 (1956) 148.
332.
R.H. Griffey, CD. Poulter, Z. Yamaizumi, S. Nishimura ['31
and B.L. Hawkins. J. Am. Chem. Sot., to be published_ 171 J. Bodenhausen and D.J. Ruben, Chem. Phys. Letters 69 (1980) 185.
PI J.D. Stoesz and A.G. Redfield. FEBS Letters 91 (1978) 320.
191 S. Alexander, Rev. Sci lnstr. 39 (1968) 1066. 1101 P. Plateau and 31. Gueron, J. Am. Chem. Sot_, to be [Ill
References
22 April 1983
published_ D.J. States, R.A. Haberkorn and DJ.
Ruben, J. Magn.
Reson. 48 (1982) 286. 1121 A.G. Redfield and SD. Kunz, in: NMR and biochemistry, eds S.J. Opella and P. Lu (Dekker. New York. 1978) p_ 125. 1131 S.D. Kunz and A.G. Redfield. Rev. Sci. lnstr.. to be publibed. I141 A.G. Redfield. J. h1ag.n. Reson., to be published.
G.
22 April 1983
CHEhlfCALPHYSICSLlXl-ERS
Volume96. number 5
TWO-DIMENSIONAL
SHIFT CORRELATION
REDFIELD
Deporrrtrenrs of Physics aladBioclzentisn-y and tlxe Rosemteil Basic Medical Sciences Research Colter. Bmndeis li’al?i!am.hinssachusetrs022.54. US.4 Received 17 January
LGversit_r.
1983
NMR spectra are obtained from Fourier transformation of the latter pti of a Hahn stimulated echo. The scqucnce is 90”-Ta-90”-qy-90”T;i-digitize ~crsus TZ_ This isdemonstrated for transfer RN.4 in Hz0 usirg the scmisclecrbc 3 bt R pulse(90”-7p-900), developed by Plateau and Cueron in place of each 90” pulse. 20 correlation between “N and protons is obtained by applying 3 90°-rt-90” l5 N sequence during q and “N dccouplin,r whifc digitizing. The second Fourier transform is performed with respect to T~ after discarding the imaginary parts of the f=st transform. to obtain a real ZD map. Such a 2D spectrum has been obtained for 5 mM *5NII,CI and several other small molecules The sequence
shouldbe useful for studiesof I5 N labeled macromo!ecules
At the time of his discovery of spitz echoes [I J Hahn also observed and analyzed s~~~?r~~~r~echoes (90”-T,-90”-lb-90°-T,-echO). Some of the recently developed two-dimensional Fourier transform (2D FT) sequences [2] are based on the concept of interference with a spin echo by irradiation on a second species. Here we extend this idea to interference with a stimulated echo_ Incidentally, it is not widely remembered that Hahn’s student, the late Bernard Herzog, performed the first double resonance heteronuclear spin echo experiment in solids years ago [3]. This was essentially a cw precursor to many modern FT techniques. Hahn and co-workers [“s] at the fBM Watson Laboratory at Columbia University also investigated the possible use of stimulated echoes for computer memory applications. A photon echo experiment qualitatively similar to what I will describe was performed by Liao et al. [S]. We developed this method in order to study a 15N labeled macromolecule, transfer RNA (tRNA). Since enviro~ental effects on “N chemical shiftsare small we need as high resolution as possible. Concentration and volume are limited to -2 mM in 0.2 ml (or less, depending on getting enough total sample), so that a proton-observe nitrogen-irradiate experiment was indicated. Griffey et al. 161 have recently labeled 0 009-2614/83/0000-OOOOjS
03.00 0 1983 North-Holland
tRNA with l5 N at the pyrimidine N3 position. and have correlated proton and nitrogen shifts by observing protons while difference-decoupling nitrogen. Resolution of 5 Hz was claimed, but in this experiment the proton lines were well resolved. If the proton lines overlap badly, interpretation will be more difficult_ Thus we sought to develop a 2D FT protonobserve experiment. The technique of Bodenhausen and Ruben 171, essentially an INEPT sequence and an inverse INEPT sequence separated by a nitrogen YO”-T, -90” sequence, should certainly be tried for this type of ap plication. However, it may suffer from some of the following problems: it requires two lS0” pulses on protons and in our probe we expected difficulty in using these clue to xf field inhomogeneity- Esperiments on tRNA must be run in Hz0 which crossrelaxes to the macromolecule protons [S], so that the spins must be allowed to recover for seconds between each sequence. Tile H20 signal would have to be saturated during the sequence to avoid overloading the spectrometer. Or else the sequence would have to be adapted for use with semiselective pulses. Finally, double iNEPT is inherently rather complicated_ The technique we have developed is tentatively called ~AHN~R, in honor of E-L_ Hahn, and the 537
CHEMICAL
Volums 96. number 5
sequence is shou II m fig. I . We first describe how a ~trmui.ued e,3lo ~eq~mx is obtained from Fourier tr~rsforrn (I’T) uf tlirz hitter part of a stimulated e&o ( t
22 April 1983
PHYSICS LETTERS
although there are amplitude variations (fig_ 1 B)- In the version of the J & R sequence that we use for transfer RNA. where the interesting part of the spectrum is far downfield, the two 90” pulse lengths are 40 ps (limited by our low power), -rp is 40 p, the frequency is =c2 kHz downfieid of solvent, and the second 90” pulse is shifted =PO” reiative to the first one. The carrier frequency is thus placed in the center of the interesting region of the spectrum (arrow). This variant of the J & R pulse was used because our proton power is rather low (3 WV). Fig_ 2 shows a single _I 8; R spectrum and a knulated echo spectra obtained in this way. on 200 nM of purified transfer RNA. Unfortunately the amplitude of the echo spectrum is decreased by a factor of about four owing to three reasons: First. a loss of 1f2 because transverse magnetization after the second pulse is lost and does not contribute to the signal. Second, a loss due to rf inhomogeneity &ich is = 1I 1.5 in our spectrometer. Third, a factor expf-2r,ifTz - rb/Tl ) due to relasation. With ra values (2”2 ms) expected to be applicable for 15N work (below) this loss will also be =l /I 5. NMR spectra have not been
j
pulse in tiu,. 1A we used ;f version of the 90”-~~-90~ pulse seq.~ence (called J & R) of Plateau and Gueron
1lo] which produces
no
phase shifts in the spectrum
I
.
I
12
14
p*m
Oownfom
*
I
.
from
\
e
10 OS
Fig_ 2. (A) i?hiR spectrum of ==l mM transfer RNA in 90%
X20, 10% DtO, pf2 6. Sample volume was 0.2 ml and the
spectrum was obtained in 53 min using a single J & R pulse. (B) Stimulated echo spectrum of the same sample using three J & R pulses as in fy. 1A. The spectrum was obtained in about one hour.
Volume96, number S
CHEMICALPHYSICS LE-iTERS
obtained in this way previously to our knowledge, and the echo spectrum may be useful for T2 measurement. Our abiIity to obtain this spectrum also shows, in our opinion, that the J & R pulse can be used to obtain types of 2D spectra in water other than that described below. The first two pulses need not be applied at the same frequency, nor have the same spacing 7 p, as the third. The main disadvantage for 2D NMR will be that since the pulse is semiselective, Tef$OnS of the 2D map will be low in sensitivity. Nowever, in certain applications where storage space or instrument time are Iimited this may be an advantage. For “N spectroscopy the sequence of fig. 1 C is added. The first interval 7a is fared and equal to slightly less than lIZA, where d is the 15NH splitting (~100 Hz). Spectra are obtained and stored for many 71 values. For reasons detailed elsewhere 12, ch. 1 ;I 11, the imaginary parts of incoming spectra obtained as described above are discarded after ET in the first dimension in order to ultimately obtain a real 2D spectrum rather than an absolute value spectrum. Doing so improves the resolution of the 2D spectrum. For each TV, four blocks of data are taken during which the second 90” l5 N pulse is sequenced through four phase shifts, by an integer JZ~times nj2_ A real echo spectrum obtained with r13 = 2 is subtracted from that with jr3 = 0 and stored. and a sirnitar difference spectrum between ?13 = 3 and 1 is stored as imaginary data for the same 71 point. The second FT is then done, with respect to I~ _ It is essential that the ls N be decoupled during digitization. These spectra were obtained on a 270 MHz system described previously Cl?], augmented by an input processor/buffer memory based on an inexpensive SlOO-bus memory [13]. Because of the limited and slow storage available (300 kbyte LINCTAF@, I developed a semitransposed storage method 1141 for the 2D FT. The output map dimensions are limited to 128 Is N by 400 proton points_ The theory of the experiment is straightforward, and will not be given. The idea is that the stimulated echo occurs if there is no change in an individual proton’s precession frequency during rt, and pdsing nitrogen will produce such a change by scrambling the nitrogen orientation for ls N protons. The time -J-, is chosen as usual so that proton isochromats are pointing oppositely for the two “IV orientations just before the second proton pulse, to maximize the effect of Is N perturbation.
“N
22 April 1983
(Coupled I
~50#HZ---4
Fig. 3. (A) Several Iincs of 3 heteronudeat 2I) experimem as shownin Fg. 1C. on SO m&lNH&l in 0.1 M NaOH.The nitrogen carrier frequency was 27 366 880 Hz. the solvent Hz0 proton resonance frequency was 270074 960 i S Hz,
and the times ~a, rP and ~1 were respectively 7 ms, 360 JXS,and O--%6 ms. The proton carrier frequency was 650 Hz downfield from H,O. (B) The PC& spectrum of the set (A) at four times biihcr gain. The noise is entirely due to spectrometer instability and is much Iessfor lines away from the proton peak, as shown in (c) which is also plotted at four times higher gain than (A).
Single lines of 22) maps for 50 mM ls NH4CI in 90% I$ 0, IO% D20 are shown in fig. 3. The unexpected appearance of the ammonium spectrum (missing central line, outer lines equal intensity) can be explained by means of a simple quantum analysis. We have not yet attempted this experiment on tRNA because the required sample has not been prepared, but I believe it will be feasible to obtain useful 2D maps at 270 MHz (5.3 T) with less than 10 mg of tRNA. Most of the noise obvious in fig_ 3B will be decreased in a labeled macromolecule. to be less than thermal noise as in fig. 3C. In principle the method may be useful for natural abundance lSN or 13C spectroscopy, but in practice it may be difficult to avoid noise due to the signal from unlabeled protons, which appears as noise because of spectrometer instability. The factor of two loss in sensitivity inherent in the stimulated echo spectrum mentioned above is theoretically not present in the double INEPT method of Bodenhausen and Ruben [7] _ The lSN resolution may be slightly better because it is the natural resolution for double INEPT but is Iimited by the proton 539
CHEMICAL
Volume 96, number 5
T1 for HAHNDOR. Nitrogen T1 can also be measured with double INEPT and not readily with HAHNDOR. For these reasons doub!e INEPT should stdl be attempted for use with labelled macromolecules despite its complexity and the probable difficultles mentioned earlier. Tills research
\\as supported
by U.S.P.H.S.
grant
GWOlbS. 1 thank Stddhartha Roy for stimulating III mterest in this subject_ Sara Kunz for expert en~~ncc:~n~ 3nd hlmnce Gueron for preliminary infornut1011 on his pulse technique.
PHYSICS
LETTERS
r41 A.G. Anderson, R-L. Garwin. E.L. Hahn. J-W_ Horton, G-L. Tucker and R-hi. WaIker, J_ AppL Phys. 26 (1955) 1324; 27 (1956) 196. ISI P-F. Liao. P. Hu, R. Leigh and S-R. Hartmann, Phys. Rev. A9 (1974)
i 1 ] K.1. II~Iln. Phys Ret. so (1950) 560. 121 A. B.Ix. T\\o JinwxGxxxi nuclcnr magnetic resonxwc xn Iq111ds (Reid& Dordrecht. 1982). [I] 1%Ilerzog_md 11.1. Iiahn. Ph?s Rev. 103 (1956) 148.
332.
R.H. Griffey, CD. Poulter, Z. Yamaizumi, S. Nishimura ['31
and B.L. Hawkins. J. Am. Chem. Sot., to be published_ 171 J. Bodenhausen and D.J. Ruben, Chem. Phys. Letters 69 (1980) 185.
PI J.D. Stoesz and A.G. Redfield. FEBS Letters 91 (1978) 320.
191 S. Alexander, Rev. Sci lnstr. 39 (1968) 1066. 1101 P. Plateau and 31. Gueron, J. Am. Chem. Sot_, to be [Ill
References
22 April 1983
published_ D.J. States, R.A. Haberkorn and DJ.
Ruben, J. Magn.
Reson. 48 (1982) 286. 1121 A.G. Redfield and SD. Kunz, in: NMR and biochemistry, eds S.J. Opella and P. Lu (Dekker. New York. 1978) p_ 125. 1131 S.D. Kunz and A.G. Redfield. Rev. Sci. lnstr.. to be publibed. I141 A.G. Redfield. J. h1ag.n. Reson., to be published.