Sensitivity improvement in three-dimensional heteronuclear correlation NMR spectroscopy

JOURNAL

OF MAGNETIC

96,4

RESONANCE

16-424

( 1992)

Sensitivity Improvement in Three-DimensionalHeteronuclear Correlation NMR Spectroscopy ARTHUR G. PALMER III, JOHN CAVANAGH, R. AND

MARK

ANDREW

BYRD,

*

RANCE

Department of Molecular Biology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, California 92037; and *Division of Biochemistry and Biophysics/CBER, Food and Drug Administration, 8800 Rockville Pike, Bethesda, Maryland 20892 Received

August

16, 199 1; revised

September

24, 199 1

Although two-dimensional proton homonuclear NMR techniques have proven successful for molecules of size up to - IO4 Da (I), larger and more complex systems are not amenable to complete study by these methods due to excessive overlap of resonances. In such cases, the larger chemical-shift dispersion of “N or r3C nuclei can be used to alleviate overlap problems (2-4). Three-dimensional heteronuclear NMR spectroscopy of isotopically enriched proteins (5,6) has been shown to be an efficient method of overcoming resonance congestion in complex protein spectra ( 7-9). Recent reports (10-12) indicate the feasibility of heteronuclear 3D NMR spectroscopy for structural investigations of polysaccharides and glycoproteins as well. In this Communication, methods are presented that provide up to a \lj: improvement in the sensitivity of NOESY-HMQC (IS-1.5), TOCSY-HMQC (16), and HSQCTOCSY experiments. The new 3D experiments are based on methods developed recently for improving the signal-to-noise-ratio in two-dimensional homonuclear isotropic mixing (TOCSY) experiments (17) and in proton-detected two-dimensional heteronuclear experiments ( 28, 19). The same pulse sequence, shown in Fig. la, is used for the conventional and the new sensitivity-enhanced (SE) TOCSY-HMQC experiments; the improvement in sensitivity is obtained by altering the manner in which the data are accumulated and processed. The principle of the method can be demonstrated for a spin system consisting of two scalar-coupled protons, A and B, with their mutual coupling constant denoted J, and a heteronucleus, S, that is scalar coupled to at least one of the protons, e.g., B, with a one-bond coupling constant JsB. Beginning with equilibrium magnetization of the A spin, evolution of coherence during the pulse sequence can be illustrated using the product-operator formalism (20-22). The effective nuclear spin Hamiltonian during the isotropic mixing period is dominated by the scalar-coupling interaction and commutes with any component of the total spin angular momentum (23). Thus, coherence transfer from proton A to proton B occurs independently during the isotropic mixing period for the two orthogonal single-spin operators present following the t, period, 0022-2364192

$3.00

Copyright 0 1992 by Academic Press, Inc. All right.9of reprcduction in any form reserved.

416

417

COMMUNICATIONS

-sgn(c#l,)A,cos wJ,cos aJ2, + -sgn( &)B,cos

aAtlcos rJt,

A,sin uAtlcos rJt, + B,sin oAtlcos ?rJt,,

[II [21

in which wA is the resonance frequency of the A spin, tl is the duration of the first incrementable time period, and sgn( r&) is the sign of the initial phase in the phase cycle for &. One-half of the number of transients recorded per increment is obtained with sgn( &) = 1, and the other half is obtained with sgn( &) = - 1 by inverting all the phases in the phase cycle for &. Multiple-spin product-operator terms have not been included in Eqs. [l] and [ 21 because these terms give rise to peaks with antiphase multiplet components that mutually cancel in large molecules with linewidths comparable to J. Concentrating on the B-proton terms, the remainder of the pulse sequence constitutes a HMQC isotope filter (24-26). The B-operator terms in Eqs. [ 1] and [ 2 ] are labeled with the chemical shift of the heteronucleus, ws, during t2; consequently the observable terms detected during the acquisition period, t3, are f [ - B,cos oAtI + B,sin uAt I ] cos 7cJt, cos ust2cos r Jt2

131

for the data acquired with sgn( &) = 1, and $[B,cos uAtl + B,sin w,tl]cos

aJt,cos uSt2cos rJt2

[41

for the data acquired with sgn( 42) = - 1. In the conventional TOCSY-HMQC experiment, the two halves of the data represented by Eqs. [ 31 and [ 41 are subtracted by inverting the phase of the receiver in synchrony with &. The recorded signal is then given by -B,cos

uAt,cos rJt,cos wst2cos aJt2.

[51

Th,e data set represented by Eq. [ 5 ] can be Fourier transformed to yield a 3D spectrum with absorptive lineshapes in all three dimensions. In the SE-TOCSY-HMQC experiment, the two halves of the data are recorded separately. Subsequently, the two data sets are alternately added to give B,sin wAtlcos aJt,cos wst2cos nJt2

[61

and subtracted to yield the same result as Eq. [ 5 1. These operations generate two new data sets that are 90” out of phase with respect to each other during the tl and t3 periods, but have the same phase during t2. The data sets can be processed to give TOCSY-HMQC spectra with pure absorption lineshapes in all dimensions and the same signal-to-noise ratio as that in the conventional spectrum. Addition of the spectra yields a 3D SE-TOCSY-HMQC spectrum in which the resonances are doubled in size, and the root-mean-square (RMS) noise is increased by a factor of fi ( 17); therefore the overall signal-to-noise ratio in the combination spectrum is improved by a factor of fi. Phase shifting and combining of the two data sets represented by Eqs. [ 5 ] and [ 6 ] to yield the final enhanced spectrum can be performed in the time or frequency domains ( 19). A similar approach can be used to obtain sensitivity enhancement of the 3D NOESY-HMQC experiment; however, modification of the conventional pulse se-

418

COMMUNICATIONS

a.

X

91

X

t2

I

C.

X

Ql

decouple

zm

z

$2

X

X

Y

X

X

t2

I

$3 II

I decouple

A dm

x

I

-Y

Y

x

Y

FIG. 1. Pulse sequences for recording three-dimensional (a) conventional and SE-TOCSY-HMQC, (b) conventional NOESY-HMQC, (c) SE-NOESY-HMQC, and (d) SE-HSQC-TOCSY experiments. The thin and thick vertical lines represent 90” and 180” pulses applied to the H (protons) or X (heteronucleus) spins; the hatched bar represents a z-gradient pulse followed by a delay to allow eddy currents to dissipate. The delay, 7, is set to 1/(2&x). Decoupling of the X spins during acquisition is accomplished using GARP1 (29) or other appropriate composite pulse sequences. Quadrature detection in the w, and wz dimensions can be achieved by the method of time proportional phase incrementation (30) or by the hypercomplex method (31, 32). Isotropic mixing is performed using the DIPSI- pulse sequence (33). The basic phase cycle for (a) is &J,= (x -x), & = x, & = (xx --x -x), and receiver = (x -x -xx). For the conventional TOCSY-HMQC experiment, the phases of +z and the receiver are inverted after four scans. For the SETOCSY-HMQC experiment, the phase of & is inverted after four scans and the two halves of the data are stored separately. The basic phase cycle for (b) is 6, = (x -x), & = (xx -x -x), and receiver = (x -x -xx). The basic phase cycle for(c) is 4, = (x -x), $z = (xx -x-x), &I~= (~JJ -y -y), and receiver = (x -x --x x). The basic phase cycle for (d) is @, = (x -x), & = (x x -x -x), 4s = ( y y -y -v), and receiver = (x --x -x x). In (c) and (d) the phase of & is inverted after four scans and the two halves of the data are stored separately.

419

COMMUNICATIONS

quence is required. Figures 1b and lc show the 3D conventional and SE-NOESYHMQC pulse sequences, respectively. The principles of the experiments can be illustrated for a system of two protons A and B which are close enough in space to crossrelax via their mutual dipolar interaction. A heteronucleus, S, again is scalar coupled to proton B. Beginning with equilibrium magnetization of the A spin, the product-operator term of interest following the tl period cross-relaxes with proton B during the mixing period to give -AA,cos uAt,cos aJt, --* -B,cos w,,,t,cos nJt,,

[71

for molecules with wo7, > 1.15, in which w. is the ‘H Larmor frequency and 7, is the rotational correlation time of the molecule. For the conventional experiment (Fig. lb), the remainder of the pulse sequence comprises an HMQC isotope filter. The final detectable operator term is - B,cos wAt, cos n-Jt , cos w& ,

[81

which yields a 3D NOESY-HMQC spectrum with pure absorptive lineshapes in all dimensions (13-15). For the SE-NOESY-HMQC sequence, the HMQC isotope filter extends to the point labeled A in Fig. lc, at which pomt the product operators present are [ -sgn( &) B,cos wSt2+ B,sin wSt2(S,cos WST+ S,sin WST)] cos wAt I cos a Jt 1.

[9]

One-half of the number of transients per t2 increment is recorded with each value of sgn ( &) . During the remainder of the sequence, the second term in Eq. [ 9 ] is refocused to a single-quantum proton operator. The resultant observable operators immediately prior to acquisition are f [ - B,COS w& - 6,,,, B ysin w&]cos wAtlcos PJt,

t101

for the data acquired with sgn( &) = 1, and 1 [B,cos wst2 - G1,NB,sin w&]cos

w,t,cos aJt,

Cl11

for the data acquired with sgn ( $2 ) = - 1. The Kronecker delta, 6, ,N, is used to indicate that the second term of Eq. [9] can be refocused only for heteronuclei with N = 1 directly attached protons (19). The two halves of the data are alternately added to yie1.d -G,,NB,cos wAtlcos HJt,sin wst2,

[I21

-B,cos wAt lcos ?rJt,cos wstz.

[I31

and subtracted to yield

The two data sets represented by Eqs. [ 121 and [ 13 ] are 90” out of phase with respect to leach other in the t2 and t3 periods and are in-phase in the tl period. As before, the two data sets can be processed to give purely absorptive lineshapes in all three dimensions. Addition of the spectra yields an overall signal-to-noise ratio increase of fi in the final enhanced spectrum for heteronuclei with single directly attached protons,

420

COMMUNICATIONS

a

j(,i I---. u IO

8,

6

4

2

wm

FIG. 2. Comparison of conventional and SE-TOCSY-HMQC spectra. A single data set was recorded on a Bruker AM600 spectrometer with the pulse sequence of Fig. la. The conventional spectrum was obtained by subtracting the two halves of the data set represented by Eqs. [3] and [4] after acquisition. A total of eight transients were recorded per (t, , tz) data point; the data matrix consisted of 256 X 64 X 2048 real points in the t, X t2 X t, dimensions. The spectral widths were 78 12 Hz in the t, and tj proton dimensions and 1506 Hz in the fz heteronuclear dimension. The isotropic mixing period was 60 ms in length. Shown are cross sections takerrfrom the 3D TOCSY-HMQC spectra parallel to the U, axes at the w2, w) frequencies of the backbone amide lSN and proton resonances for the spin systems of (a) Lys 12 and (b) Glu 35. The top and bottom traces show the sensitivity-enhanced and conventional spectra, respectively. The increase in the RMS baseplane noise for the enhanced spectrum, compared to the conventional one, agrees with the theoretical value of fi. The average signal-to-noise improvements between the enhanced and conventional spectra are (a) 1.42 and(b) 1.38.

compared to the conventional NOESY-HMQC experiment. As has been described in detail elsewhere (19), the actual improvement in the signal-to-noise ratio in the 3D SE-NOESY-HMQC experiment will be somewhat less than fi, due to relaxation during the longer pulse sequence of Fig. lc. Three-dimensional experiments with enhanced sensitivity also can utilize the HSQC heteronuclear hlter; for example, the sequence for a 3D SE-HSQC-TOCSY experiment is shown in Fig. Id. In this experiment, the enhanced HSQC sequence previously described ( 19) is used as the isotope filter; consequently, the product-operator analysis of the sequence parallels the analysis presented above for the SE-HMQC-NOESY experiment. In particular, one of the two orthogonal magnetization components recorded in the 3D SE-HSQC-TOCSY experiment will be filtered according to the number of protons attached to the heteronuclei (19). The above theoretical analyses were verified experimentally by recording ‘H- “N‘H 3D conventional and sensitivity-enhanced TOCSY-HMQC and NOESY-HMQC

421

COMMUNICATIONS

1 10

I 8

1 6

I 4 wm

I 2

IO

6

6

4

2

0

ppm

FIG. 3. Comparison of conventional and SE-NOESY-HMQC spectra. The experiments were performed on a Bruker ANX-500 spectrometer using the pulse sequences of Figs. lb and lc. A total of 16 transients were recorded per (I,, t2) data point; the data matrix consisted of 100 complex X 32 complex X 2048 real points in the t, X tz X t, dimensions, respectively. The spectral widths were 7042 Hz in the t, and tSproton dimensions and 1250 Hz in the t, heteronuclear dimension. The NOESY mixing time was 150 ms. Both experiments were recorded in the same overall time. Shown are cross sections for (a) Lys 12 and (b) Glu 35 t&ken from the 3D NOESY-HMQC spectra parallel to the w, axes at the w2, wg frequencies of the backbone amide 15N and proton resonances, respectively. The top and bottom traces show the sensitivityenhanced and conventional spectra, respectively. The increase in the RMS baseplane noise for the enhanced spectrum, compared to the conventional one, agrees with the theoretical value of fi. The average signalto-noise improvements between the enhanced and conventional spectra are (a) 1.4 1 and (b) 1.39.

spectra using the pulse sequences given in Figs. la- lc, and by recording a ‘H- 13C‘H SE-HSQC-TOCSY spectrum using the pulse sequence of Fig. Id. TOCSY-HMQC and NOESY-HMQC experiments were performed on a 2.5 mM solution of calbindin Dgk (27) in 90% HzO/ 10% D20 at 300 K. Calbindin Dgk is a 75amino-acid calciumbinding protein; the sample used for NMR experiments was uniformly labeled with 15N to >95%. The SE-HSQC-TOCSY experiment was performed on a 1.3 mMsolution of the Kl antigen polysaccharide in D20 at 328 K. The Kl antigen was isolated from Escherichia coed(28) and was uniformly r3C enriched to 33%. Additional experimental details are given in the legends to Figs. 2-4. Examples of the processed 3D spectra obtained for the TOCSY-HMQC, NOESYHMQC, and HSQC-TOCSY experiments are given in Figs. 2, 3, and 4, respectively. As discussed in the legends, in each case the sensitivity improvement afforded by the enhanced experiment compared to the conventional experiment agreed with the theoretical factor of \/2. Figure 2 ilhtstrates the sensitivity improvement obtained for TOCSY transfer through the spin systems of Lys 12 and Glu 35 in calbindin Dgk.

422

COMMUNICATIONS

4.5

4.0

3.5

3.0

2.5

2.0

1.5

PPm

FIG. 4. SE-HSQC-TOCSY spectra of Kl polysaccharide. The spectrum was recorded on a JEOL GSX500 spectrometer using the pulse sequence of Fig. Id. A total of 64 transients were recorded per (t, , tz) complex data point, and the data matrix consisted of 60 X 32 X 256 complex points in the t, X tz X ts dimensions. The spectral widths were 1667 Hz in the t, and t, proton dimensions and 3571 Hz in the tz carbon dimension. Extensive folding of the spectrum and linear prediction were utilized in the carbon dimension (34,35) to achieve acceptable digital resolution. The isotropic mixing period was 22 ms. Longer isotropic mixing is prohibited by the short r,, of the protons in this polymer (effective molecular weight of ca. 30-50 kDa). Shown are cross sections parallel to the ws axis at the w2 chemical shift that contains both C4 (7 1.O ppm) and C3 (42.6 ppm) resonances as a result of folding in w2. The proton w, chemical shifts are (a) 3.75 ppm corresponding to H4, (b) 2.69 ppm corresponding to H3e, and (c) 1.78 ppm corresponding to H3a. The folded resonances are inverted relative to resonances that have not been folded. The sensitivity gain in this experiment affords excellent correlations for magnetization that originates on CH spin systems (a). The TOCSY peaks shown in (b) and (c) are expected to exhibit decreased signal-to-noise ratios because the magnetization originates with CH2 spin systems (19). The expected gain in signal-to-noise ratio has been independently verified for the two-dimensional version of this sequence.

Figure 3 illustrates the sensitivity improvement obtained for magnetization transfer via cross-relaxation with the amide proton of the spin systems of Lys 12 and Glu 35 in calbindin Dgk. Figure 4 demonstrates the TOCSY transfer from the H4, H3e, and H3a protons of the Kl antigen polysaccharide. To conclude, two approaches for increasing the sensitivity of 3D heteronuclear NMR experiments have been introduced. Although the experimental demonstrations of the new methods have utilized the HMQC and HSQC isotope filters, the sensitivityenhancement procedure described here can be implemented with other heteronuclear filters (19) or different permutations of the proton and heteronuclear evolution periods, e.g., NOESY /TOCSY-HSQC. In the present examples, the overall gains in sensitivity obtained with the new methods agree well with the theoretical maximum of fi. The

COMMUNICATIONS

423

pulse sequence for the SE-TOCSY-HMQC experiment is identical to that of the conventional experiment; therefore, relaxation does not affect the obtainable sensitivity enhancement. Sensitivity is improved in the NOESY-HMQC and 3D HSQC-TOCSY experiments by recording magnetization arising from two orthogonal heteronuclear spin operators. The pulse sequences required are slightly longer than the conventional sequence; consequently additional losses of signal due to relaxation processes may occur, and improvements in signal-to-noise ratios of less than e generally will be obtained. Sensitivity enhancement is most strongly affected by relaxation in experiments that use the HMQC isotope filter (19); consequently, for proteins larger than calbindin, the SE-NOESY-HSQC experiment may be preferable to the SE-NOESYHMQC experiment. ACKNOWLEDGMENTS This work was supported by a grant from the National Science Foundation (DMB 8903777). J.C. thanks The Royal Society for the award of the Foulerton Gift and Binmore Kenner Research Fellowship. A.G.P. was supported by a National Science Foundation Postdoctoral Fellowship in Chemistry awarded in 1989 (CHE-8907510). We thank Drs. W. J. Chazin (Scripps) and E. Thulin (Lund) for the isotopically labeled calbindin Dgk. Dr. N. J. Skelton (Scripps) for discussions concerning calbindin assignments, and Dr. W. F. Vann (CBER/FDA) for the isotopically labeled Kl polysaccharide. REFERENCES 1. K. WORTHRICH, “NMR of Proteins and Nucleic Acids,” Wiley, New York, 1986. 2. A. BAX, S. W. SPARKS, AND D. A. TORCHIA, “Methods in Enzymology” (N. Oppenheimer and T. L. James, Eds.), Vol. 176, pp. 134-150, Academic Press, San Diego, 1989. 3. G. WAGNER, “Methods in Enzymology” (N. Oppenheimer and T. L. James, Eds.), Vol. 176, pp. 93I 14, Academic Press, San Diego, 1989. 4. R. H. GRIFFEY AND A. G. REDFIELD, Q. Rev. Biuphys. 19, 51 ( 1987). 5. L. P. MCINTOSH, R. H. GRIFFEY, D. C. MUCHMORE, C. P. NIELSON, A. G. REDFIELD, AND F. W. DAHLQUIST, Proc. Nat/. Acud. Sci. USA 84, 1244 ( 1987). 6. L. P. MCINTOSH AND F. W. DAHLQUIST, Q. Rev. Biophys. 23, 1 ( 1990). 7. L. E. KAY, D. MARION, AND A. BAX, J. Magn. Reson. 84,12 (1989). 8. L. E. KAY, M. IKURA, R. TSCHUDIN, AND A. BAX, J. Magn. Reson. 89,496 ( 1990). Y. S. W. FESIK AND E. R. P. ZUIDERWEG, Q. Rev. Biophys. 23,97 ( 1990). 10. R. A. BYRD, S. FREESE, AND W. F. VANN, 32nd ENC, St. Louis, Missouri, April 7-11, 1991. II. S. W. FESIK, R. T. GAMPE, JR., AND E. R. P. ZUIDERWEG, J. Am. Chem. Sot. 111,770 ( 1989). 12. P. DEWAARD, R. BOELENS, G. W. VUISTER, AND J. F. G. VLIEGENHART, J. Am. Chem. Sot. 112,3232 (1990). 13. S. W. FESIK AND E. R. P. ZUIDERWEG, J. Magn. Reson. 78,588 ( 1988). 14. D. MARION. L. E. KAY, S. W. SPARKS, D. A. TORCHIA, AND A. BAX, J. Am. Chem. Sot. 111, 15 I5 (1989). 15. E. R. P. ZUIDERWEG AND S. W. FESIK, Biochemistry 28,2381(1989). 16. D. MARION, P. C. DRISCOLL, L. E. KAY, P. T. WINGFIELD, A. BAX, A. M. GRONENBORN, AND G. M. CLORE, Biochemistry 28,6 150 ( 1989). 17. J. CAVANAGH AND M. RANCE, J. Magn. Reson. 88,72 ( 1990). 18. J. CAVANAGH, A. G. PALMER, P. E. WRIGHT, AND M. RANCE, J. Magn. Reson. 91,429 ( 1991). 19. A. G. PALMER, J. CAVANAGH, P. E. WRIGHT, AND M. RANCE, J. Magn. Reson. 93, 15 1 ( 1991). 20. 0. W. %RENSEN, G. EICH, M. H. LEVITT, G. BODENHAUSEN, AND R. R. ERNST, Prog. NMR Spectrosc. 16, 163 (1983). 21. K. J. PACKER AND K. M. WRIGHT, Mol. Phys. 50,797 ( 1983). 22. F. J. M. VAN DE VEN AND C. W. HILBERS, J. Mugn. Reson. 54,5 12 ( 1983).

424

COMMUNICATIONS

23. L. BRAUNSCHWEILER AND R. R. ERNST, J. Magn. Reson. 53,521 ( 1983). 24. L. MULLER, J. Am. Chem. Sot. 101,160 (1979). 25. M. R. BENDALL, D. T. Peck, AND D. M. D~DDRELL, J. Magn. Reson. 52,8 1 ( 1983 ). 26. A. BAX, R. H. GRIFFEY, AND B. L. HAWKINS, J. Magn. Reson. 55,301 (1983). 27. J. KORDEL, S. FORSEN, AND W. J. CHAZIN, Biochemistry 28,7065 ( 1989). 28. A. K. BHATTACHARJEE, H. J. JENNINGS, C. P. KENNY, A. MARTIN, AND I. C. P. SMITH, J.

250, 1926 (1975). 29. A. J. SHAKA, P. B. BARKER, AND R. FREEMAN, .I. Magn. Reson. 64,547 ( 1985). 30. D. MARION AND K. WOTHRICH, Biochem. Biophys. Res. Commun. 113,967 ( 1983). 31. L. MULLER AND R. R. ERNST, Mol. Phys. 38,963 ( 1979). 32. J. KEELER AND D. NEUHAUS, J. Magn. Reson. 63,454 ( 1985). 33. A. J. SHAKA, C. J. LEE, AND A. PINES, J. Magn. Reson. 77,274 (1988). 34. A. BAX, M. IKURA, L. E. KAY, AND G. ZHU, J. Magn. Reson. 91, 174 ( 1991). 35. G. ZHU AND A. BAX, J. Magn. Reson. 90,405 ( 1990).

Biol. Chem