Multiple-quantum 113Cd1H correlation spectroscopy as a probe of metal coordination environments in metalloproteins

JOURNAL

OF MAGNETIC

RESONANCE

61, 579-584

(1985)

Multiple-Quantum l13Cd-‘H Correlation Spectroscopyas a Probe of Metal Coordination Environments in Metalloproteins JAMES D. OTVOS, HELEN R. ENGESETH, AND SUZANNE WEHRLI Department of Chemistry, University of Wisconsin, Milwaukee, Wisconsin 53201 Received November 6, 1984

The utility of ‘13Cd NMR as a structural probe of biological metal coordination sites has been amply demonstrated in recent years (I, 2). By monitoring the chemical shifts and relaxation properties of the resonances from ‘13Cd2+ substituted for the native metal-ion cofactors of metalloproteins, it is possible to infer the identities of the protein ligands to the metal, the strength of binding, and the rates of functionally significant ligand exchange reactions. Scalar coupling of the spin-i ‘13Cd nucleus to NMR-detectable nuclei of its protein ligands can also be usefully exploited for complementary structural information provided that the resonances of these nuclei are sufficiently well resolved. Unfortunately, this requirement is rarely met even in relatively small proteins since the ‘H and 13C signals of the metal-ion ligands are usually obscured by overlap with other resonances. Only when proteins have been investigated that contain 13C-enriched ligands (3) or less abundant ligand nuclei such as 3’P (4-6) has this potential source of information been accessible. In this paper we demonstrate the feasibility of using multiple-quantum NMR to obtain ‘H NMR spectra of a ‘13Cd protein in which the only resonances to appear are from protons spin coupled to cadmium. The pulse sequence used is one which has been recently shown to have great potential for the indirect detection of lowsensitivity nuclei such as “N and the generation of superior heteronuclear chemicalshift correlation maps of peptides and nucleic acids (7, 8). Since sensitivity is expected to suffer when the multiple-quantum coherences are generated by relatively small scalar couplings (because of transverse relaxation during the relatively long preparation period), we were concerned that reliance on long-range ‘13Cd-‘H coupling (through three or more bonds) would produce data that were far less useful than those obtained by others using one-bond “N-‘H couplings. The system we chose to test this possibility was metallothionein, a 6800 MW protein that contains seven cadmium ions arranged in two metal-thiolate clusters via coordination to 20 cysteine residues (9). Despite the fact that the P-protons of the cysteine ligands exhibit a wide range of coupling constants (3Jcd-u - O-70 Hz) the multiplequantum experiment was found to give good sensitivity and permit many of the ligand protons to be correlated with the ‘13Cd ion(s) to which they are spin coupled. The pulse sequence used to obtain the multiple-quantum ‘13Cd-‘H chemical shift

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Copyright 0 1985 by Academic Press. Inc. All rights of reproductmn in any form reserved

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correlated spectrum was that described by Bax et al. (sequence Id in Ref. (7)) and Bendall et al. (8) and is as follows: I ‘H: I n/2(x) 44 AQ

1’3Cd:

[ --a---?r/2(~k--t,,r- ; -+--~/2(x)I

The delay A is set to maximize the antiphase components of magnetization in the transverse plane prior to their conversion into multiple-quantum coherence by the first ‘13Cd pulse. Thus, for an H-X doublet arising from a one-bond coupling, A is normally set to 1/2J (7, 8). In metallothionein, a compromise value of A must be used because there is a wide range of JHxd values, some protons are coupled to more than one ’ r3Cd ion, and transverse relaxation during A must be taken into account. We have used A = 30 ms for the experiments reported here. The evolution period t, was varied from 0 to 16 ms in sixty-four 0.25 ms steps to give the data in Fig. 1B or in one hundred twenty-eight 0.125 ms steps for the spectrum in Fig. 2 to give a spectral width in the r13Cd dimension of 4000 Hz. The phase, 4, of the first ‘13Cd 90” pulse was varied in four steps along with the receiver phase to select the zero-quantum coherence and cancel all signals from protons not coupled to r 13Cd (7). The phases, x, of the other pulses were stepped together in intervals of 90” in groups of four to give a sequence consisting of a total of 16 pulses. All experiments were performed at 250 MHz on a Bruker WM-250 spectrometer using the decoupler coil of a broadbanded probe to detect the ‘H signals and the tunable observe coil to transmit the ‘r3Cd pulses. The ‘13Cd frequency (55.5 MHz)

5

4

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1

PPrn FIG. 1. (A) ‘H NMR spectrum of “‘Cd,-rabbit liver metahothionein-2 (500 transients). Resolution was enhanced by use of a Lorentzian-to-Gaussian transformation. The sharp, intense peaks at 3.81 and 4.75 ppm are from residual Tris buffer and HDO, respectively. (B) The absolute-value-mode projection onto the ‘H axis of a two-dimensional “3Cd-‘H correlated spectrum obtained on the same protein sample. The ‘H spectral width was 1500 Hz and 5 12 transients were accumulated for each value of tl , giving a total acquisition time of ca. 16 h.

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n

-630 ‘13Cd ( PPm)

-650

I I

-670

I

I I

I

4

5 1

H

I

1

3

(PPrn)

FIG. 2. A “‘Cd-‘H chemical shift correlation spectrum of “SCd,-metallothionein-2. The ‘H spectral width was 800 Hz and 720 transients were accumulated for each value of t,, giving a total acquisition time of ca. 45 h. Along the ‘H axis at the top of the spectrum is plotted the absolute-value-mode projection. Along the “‘Cd axis on the left side of the spectrum is a proton-decoupled ‘13Cd spectrum of the same sample. The labeling of the peaks (I-VII) corresponds to the designations given to the metals in the model of the metal-thiolate clusters in Ref. (9). The contours at 3.8 1 and 4.75 in the two-dimensional spectrum arise from the incompletely suppressed residual Tris buffer and HDO signals.

was generated by a synthesizer and amplified, phase shifted, and gated by a Bruker BSV-3 decoupling unit. The ‘13Cd 90” pulse width was determined to be 23 ps using a ‘i3Cd-EDTA test sample and the calibration method devised by Pegg et al. (10) and recently described by Bax (II). Measurements were made at 42°C using 10 mm NMR tubes. ’ i3Cd chemical shifts are referenced to external 0.1 M Cd(ClO& and proton shifts to TSP as usual. The metallothionein was isolated from the livers of rabbits subjected to repeated injections of Zn(SO& using established procedures (9). The pure Zn7-Mt-2 isoprotein was converted to ’ 13Cd7-Mt-2 by direct displacement of the Zn*+ by ‘i3Cd2+ (95% enriched isotope from Prochem). The protein was dissolved in 99.9% D20 at a concentration of 6.9 mM in 50 nGt4 sodium phosphate buffer, pH 8.0. Despite having only 61 amino acid residues, metallothionein gives rise to a complex ‘H NMR spectrum at 250 MHz (Fig. 1A). Few resolved resonances attributable to individual protons are observed because the entire spectrum is confined to a narrow chemical-shift range of ca. l-5 ppm. In particular, the

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resonances of the 40 P-CH2 protons of the 20 metal-coordinating cysteine residues in the region around 3 ppm, which could potentially be utilized as probes of the local environments of the two metal-thiolate clusters, are largely obscured by the signals of 8 lysine, 9 serine, and 3 aspartic acid residues (12) which appear in the same region. In marked contrast is the spectrum in Fig. lB, which was derived from the two-dimensional data set of a multiple-quantum ‘13Cd-‘H shift correlation experiment performed on the same protein sample. Here, the suppression of resonances from protons not coupled to ‘13Cd is virtually complete, leaving only the cysteine P-CH2 proton resonances between 2.6 and 4.0 ppm. The signal at 4.75 ppm represents incompletely suppressed HDO. The 5.25 ppm resonance is not an artifact but arises from another “3Cd-coupled proton, though apparently not a cysteine /3-CH2 proton. We have tentatively assigned it to a cysteine (Y-CH on the basis of its low field shift, long T2, ‘H-‘H coupling connectivity, and small J,,‘Cd-,H (-6 Hz). Although the spectrum in Fig. 1B is greatly simplified owing to the absence of resonances from protons not coupled to ‘13Cd, it is still complex because of the presence of both ‘H-‘H and ‘H-“3Cd coupling as well as the superpositioning of resonances from protons coupled to different i’3Cd2i ions in the protein. The latter complexity is removed when the full ‘H-‘13Cd shift correlation spectrum is plotted, as in Fig. 2. Here, the protons coupled to each of the ‘13Cd2’ ions in the threemetal cluster (peaks II-IV) and four-metal cluster (peaks I and V-VII) are clearly separated from one another in the ‘13Cd dimension. The prevailing model of the metal clusters in metallothionein (9) predicts that eight cysteine &CH2 protons should be coupled to each of the seven ‘13Cd2’ ions and that the protons of the eight bridging thiolate ligands should be additionally coupled to a second “3Cd2+. Clearly, not all of these protons are represented in the two-dimensional spectrum in Fig. 2, although most of those coupled to cadmiums I and V do appear. The missing proton resonances are not detected because their spin couplings to cadmium are too small and/or their transverse relaxation is so efficient that little or no signal remains during the detection period. To some extent, we have found this problem can be alleviated by acquiring data with several different delay periods, A. However, protons with very small couplings (~4 Hz) will probably always be undetectable. Assuming a Karplus-type relationship exists for three-bond ‘13Cd-‘H coupling, it is understandable that many cysteine proton resonances are not observed because one member of a P-CH2 pair would be predicted to have a small coupling to ‘13Cd when the other’s coupling is large. Unlike metallothionein, most metal-binding proteins that might be usefully investigated by ‘13Cd-‘H multiple-quantum NMR contain only one or two different metal coordination sites. In these cases there would be no need to collect the full two-dimensional data set if the ‘13Cd chemical-shift information was not required. Instead, it would be more efficient to acquire data using a short, fixed delay period, tl , and nonselective ‘13Cd pulses to obtain in a short period of time a onedimensional spectrum containing information equivalent to that found in the ‘H projection (such as in Fig. 1B) of a full two-dimensional experiment. For proteins whose “‘Cd NMR spectrum contains more than one resonance, the same procedure using selective ‘13Cd pulses would give information equivalent to slices through the

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two-dimensional spectrum at the ‘13Cd frequency of the irradiated resonance. Such an experiment was successfully tested on metallothionein, as shown in Fig. 3. All of the /3-CH2 resonances which appear in the two-dimensional spectrum in Fig. 2 are found in the one-dimensional zero-quantum spectrum in Fig. 3A obtained with nonselective ’ 13Cd pulses. In Figs. 3B and C are the “subspectra” obtained with selective ‘13Cd pulses app lied to resonances V and VII, respectively. The multiple-quantum technique described here is only one of many possible methods by which 113Cd-1H spin couplings may be exploited to suppress noncoupled proton resonances or provide ‘13Cd-‘H chemical shift correlation. Of those we have tried, however, it appears to give by far the best results. One-dimensional techniques such as double-resonance difference spectroscopy (13) and spin-echo difference spectroscopy (14) using either selective or nonselective ‘13Cd pulses have the potential advantage of requiring shorter data acquisition times, but at least in our hands give inferior cancellation of noncoupled signals due to their greater sensitivity to instrumental instabilities and their reliance on a relatively narrow range of H-X coupling constants. Conventional heteronuclear chemical shift correlation spectroscopy (15) using ’ 13Cd detection has the potential advantage of avoiding the need to suppress noncoupled proton signals, but gives greatly inferior sensitivity and provides in practice lower digital resolution in the ‘H dimension where it is most needed. Based on our results with metallothionein we believe multiple-quantum ‘13Cd-‘H

3.6

3.2

2.8

PPm

obtained with FIG. 3. One-dimensional zeroquantum ‘H NMR spectra of “3Cd7-metallothionein-2 the same pulse sequence used in Figs. 1 and 2 except that the r(x) proton pulse was omitted. A fixed t, period of 20 e was used with A = 30 ms. 5000 transients were accumulated for each spectrum, requiring 2.5 h each. Spectrum A was obtained using nonselective (23 11s) “‘Cd pulses and spectra B and C were pulses applied at the frequencies of resonances V and VII, obtained using selective (3.1 ms) “3Cd respectively. All three spectra are in the absolute-value mode.

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NMR in both its one- and two-dimensional versions will become a valuable new source of structural information on metal-binding proteins which will complement and extend that which is already accessible via ‘13Cd NMR. ACKNOWLEDGMENT This

work

was supported

by National

Institutes

of Health

Grant

GM

29583.

REFERENCES 1. I. M. ARMITAGE AND J. D. OTVOS, “Biological Magnetic Resonance” (L. J. Berliner and J. Reuben, Eds.), Vol. 4, pp. 79-144, Plenum, New York, 1982. 2. I. M. ARMITAGE AND Y. BOULANGER, “NMR of Newly Accessible Nuclei,” Vol. 2, pp. 337-365, Academic Press, New York, 1983. S. J. D. OTVOS AND I. M. ARMITAGE, Biochemistry 19, 4021 (1980). 4. J. D. OTVOS, J. R. AU;ER, J. E. COLEMAN, AND I. M. ARMITAGE, J. Biol. Chem. 254, 1778 (1979). 5. G. I. RHYU, W. J. RAY, JR., AND J. L. MARKLEY, Biochemistry 23, 252 (1984). 6. K. M. WELSH, I. M. ARMITAGE, AND B. S. COOPERMAN, Biochemistry 22, 1046 (1983). 7. A. BAX, R. H. GRIFFEY, AND B. L. HAWKINS, J. Magn. Reson. 55, 301 (1983). 8. M. R. BENDALL, D. T. PEGG, AND D. M. DODDRELL, J. Magn. Reson. 52, 81 (1983). 9. J. D. OTVOS AND I. M. ARMITAGE, Proc. Natl. Acad. Sci. USA 77, 7094 (1980). 10. D. T. PEGG, M. R. BENDALL, AND D. N. DODDRELL, J. Magn. Reson. 44,238 (198 1). Il. A. BAX, J. Magn. Reson. 52, 76 (1983). 12. M. KIMURA, N. OTAKI, AND M. IMANO, “Metallothionein” (J. H. R. Kagi and M. Nordberg, Eds.), pp. 163-168, Birkhauser Verlag, Basel, 1979. 13. J. P. MARCHAND AND D. CANET, J. Am. Chem. Sot. 97,658l (1975). 14. R. FREEMAN, T. H. MARECI, AND G. A. MORRIS, J. Magn. Reson. 42, 341 (1981). 15. G. BODENHLJSEN AND R. FREEMAN, J. Magn. Reson. 28,471 (1977).