Magic-angle-sample-spinning NMR difference spectroscopy

JOURNAL

OF MAGNETIC

RESONANCE

77,25 l-257 (1988)

Magic-Angle-Sample-Spinning

NMR Difference Spectroscopy

H. J. M. DE GROOT,* V. COPIB,*T~ S. 0. SMITH,* P. J. ALLEN,* C. WINKEL,~ J.LUGTENBURG,$J.HERZFELD,§ANDR.G.GRIFFIN* *Francis Bitter National Magnet Laboratory and fDepartment of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; $Gorlaeus Laboratories, Rijksuniversiteit te Leiden, P.O. Box 9502, 2300 RA Leiden, The Netherlands: and $Department of Chemistry, Brandeis University, Waltham, Massachusetts 02254 Received June 17, 1987; revised August 24, 1987 Magic-a&e-sample-spinning (MASS) NMR difference spectra can lx used to selectively observe the contribution of an isotopic label to the total NMR spectrum. Because the natural abundance background is removed, the measurement of centerband and sideband intensities, and therefore chemical-shift anisotropies, is facilitated. This is illustrated with difference spectra between native and specifically labeled bacteriorhodopsin. A device which permits precise setting and regulation of the spinning speed is required for these experiments and is described herein. The apparatus should also be useful in other MASS experiments where knowledge and regulation of the spinning speed is reqmred. Q 1988 Academic FWSS.IIIC. INTRODUCTION

During the past few years there has been considerable progress in the development of sophisticated techniques for observation of high-resolution nuclear magnetic resonance spectra of dilute nuclei in the solid state (1, 2). One of the most frequently utilized approaches involves magic-angle-sample spinning (MASS) (3), often in combination with cross polarization and dipolar decoupling (4, 5). It is well known that at low fields and/or at high spinning speeds MASS NMR spectra resemble those observed in liquids in that they exhibit a single line for each magnetically inequivalent nucleus. In contrast, in the slow spinning regime rotational sidebands, spaced at multiples of the spinning speed frequency (vn), appear in MASS spectra, in addition to the centerbands at the isotropic chemical shift. Information on the chemical-shift anisotropy can be obtained from the sideband intensities (6) and such data have been exceptionally useful in understanding structural and electronic changes which are usually manifested only as changes in the isotropic chemical shift. At the same time the presence of multiple sidebands can lead to spectral overlap which interferes with the accurate measurement of sideband intensities. In certain cases of interest, a relatively straightforward solution to this problem is possible using difference spectroscopy. For example, it was shown some time ago that dipolar suppression methods could be employed to attenuate the 13C resonances of protonated carbons. Subsequent application of difference techniques, where the unprotonated spectrum is subtracted from the complete spectrum, generates a spectrum containing lines from only the protonated 13C’s (7). More recently, we have been studying spectra of large biological molecules which are isotopically labeled with, among 251

0022-2364188 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

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others, 13Cand “N. In this case it is possible to record the spectrum of the isotopically labeled and natural abundance (native) samples, and their dif?&ence yields the spectrum of the label alone. Since the difference spectrum contains lines from only one, or at most a few sites, it is easy to measure the sideband intensities and to determine the shift anisotropies. The purpose of this paper is to demonstrate the feasibility of MASS NMR difference spectroscopy. In order to obtain MASS NMR difference spectra which are free of artifacts and accurately reflect the spectrum of the label, it is necessary to record spectra under as nearly identical conditions as possible. An important variable in this process is the spinning speed, which must be identical in the two spectra. In order to ensure a constant spinning speed, we have constructed a device based on a mass flow controller which permits the speed to be set precisely to a desired value, and to be maintained to within 2 Hz of the chosen set point. We demonstrate the utility of this approach with a measurement of the shift anisotropies for a i3C label in a large membrane protein-[ 14-‘3C]retinal in the dark-adapted form of bacteriorhodopsin (bR). In addition to the work reported here at least two other groups have performed MASS NMR difference spectroscopy (8). In one case (8~) a controller was not employed and in the second case (8b) only the isotropic resonance was of interest. Comparison of the present results with the other work clearly shows the improvement that may be achieved by following procedures outlined here. THE

SPINNING

SPEED

CONTROLLER

Recently, a device for measuring and controlling the spinning speed during a MASS NMR experiment has been described by Lee et al. (9). They developed a technically elegant apparatus, which detects the spinning speed optically, generates an error signal proportional to the deviation from the value of VRset by the experimenter, and fmaIly adjusts the gas flow by means of a precision needle valve controlled by a stepping motor. The spinning speed controller we have constructed has evolved from this earIier design. However, we have modified the apparatus extensively and therefore a separate discussion is in order. The block diagram of our apparatus is given in Fig. 1, The modifications are (i) replacement of the stepping motor and needle valve by a mass flow controller; (ii) improvement in sensitivity of the optical detection system; (iii) extension of the control range; (iv) improvement in precision of the spinning speed setting by two digits; and (v) addition of two control adjustments. Probably the most important modification of the apparatus is the first since it considerably improves the performance of the system at its most delicate point, the regulation of the gas flow. There were several reasons for replacing the stepping motor and needle valve. First, the range over which the gas Ilow could be adjusted was quite limited, corresponding to an interval of at most 10 Hz around the set point fur our spinners. According to the original design the controller disabled itself automatically whenever the valve reached this limit, and therefore fairly minor disturbances in the gas flow, e.g., due to small leaks in the tubing, were enough to interrupt the NMR experiment. Second, the construction of a reliable stepping motor/valve system is difficult and time-consuming. Finally, the original valve system did not incorporate

MAGIC-ANGLE-SPINNING

DIFFERENCE

pressure repu,ator

SPECTROSCOPY

253

pressure regulator ,I,

FIG. 1. Block diagram of the spinning speed controller, showing the electronic and the pneumatic components. Gas conduits are denoted by shaded lines. Note that as in the original design a precision pressure regulator (I 7) is included upstream of the mass flow controller. A computer tape with wire-wrap instructions for the electronic part is available upon request.

the measurement of the actual gas flow through the spinner. Although the latter is not strictly necessary for the NMR experiment, it is nevertheless very useful when troubleshooting is required or when comparing the performances of different spinners and rotors. The incorporation of the mass flow controller (10) eliminates these problems. It allows continuous regulation and measurement of the gas flow from zero up to 50 liters per minute with an absolute accuracy of 10 ml per minute. This corresponds to an absolute error of - k2 Hz in the spinning speed, which is excellent considering that the linewidths typically observed in high-resolution solid-state NMR spectra are of the order of 30-40 Hz. In fact, the mass flow controller almost entirely determines the stability of VR. The spinning speed controller corrects for the long-term drift by adjustment of this stable gas flow. The input signal for the mass flow controller is generated with a digital-to-analog converter (DAC) and an up/down counter. The error pulse train is used either to increase or to decrease the counter value, depending on the sign of the error, as indicated by the direction bit. Each time the counter is assigned a new value, it is read by the DAC and the input signal for the mass flow controller is adjusted. The correction goes in steps of 12 ml per minute, which is approximately equal to the absolute error of the mass flow controller. In order to keep the feedback loop for the spinning speed controller short, the mass flow controller has been mounted as close to the probe as possible, taking into account that proper operation of the device will be disturbed by a strong magnetic field. In order to provide sufficient light intensity for the photodetection of the spinning speed from the half-white, half-black painted bottom of the rotor, we initially employed a He-Ne laser. Presumably, this was required because the surface area on the bottom

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of our rotors is much smaller than the area of the T-barrel rotor developed and used by Lee et al. Modification (ii), which consists of a two-stage amplifier and band-pass filter, enabled us to use a considerably less expensive light source. At present we employ a No. 328 incandescent light bulb operating at its regular 5 V supply voltage. The filter consists of an active high-pass filter with a cutoff frequency of 100 Hz and an active low-pass filter with a cutoff frequency of 7.5 kHz. The amplifier gain in the passband amounts to 20 dB. The filter also removes the dc offset from the photodiode signal and it strongly suppresses the noise. In addition we have modified the input stage circuitry so that the comparator now has a constant hysteresis of - 100 mV. To transmit the light into the probe and back to the detector various dif&ent types of fiber optic cable are used, both custom-manufactured (II) and homemade from relatively inexpensive bundle fiber (12), all of which have worked satisfactorily. Wherever necessary SMA-type fiber optic connectors and receptacles are installed. For the polishing of the fibers we use the standard accessories provided by the manufacturer of the connectors. The purpose of modification (iii) is to increase the control range of the device. Instead of an 8-bit counter we have used a 12-bit device, yielding an increase of the control range to -~4% ofthe actual set value for the spinning speed (formerly ?0.25%). Modification (iv) has been carried out according to the guidelines given by Lee et al. (9) and allows the spinning speed to be set in multiples of one hertz. The last modification, (v), provides the experimenter with two controls for optimization of the performance of the apparatus. First of all, we added a programmable divider (-+lw) that varies the repetition frequency for the correction actions. It replaced the divide-byfour circuit in the original design. The main reason for this modification is that the response time of the spinner assembly on changes in the airflow depends considerably on VR. The lower the VR, the longer iS this response time and this may @Ve rise to oscillatory behavior of VRat the lowest frequencies. The second control is a divider in the pulse train (t.L), which allows for attenuation of the error signal. It should he adjusted for maximum possible feedback, i.e.. in such a way that the system is just below the oscillation point. With this spinning speed controller we are able to control speeds from -500 Hz up to the highest frequencies that may be obtained with the spinners and rotors. The system appears to be very stable, and thus far it has not lost control during any experiment. NMR

DIFFERENCE

SPECTRA

OF

BACTERIORHODOPSIN

Bacteriorhodopsin is a light-harvesting protein with a retinylidene chromophore at its active site (13). It has been found to act as a light-driven proton pump (14) and during the past few years a considerable effort has been devoted to understanding its mechanism. Very recently, MASS NMR techniques have: been applied to examine both the microscopic structure of the retinal chromophore and the protein charge distributions around this chromophore (15,16). In Fig. 2A we show 13C MASS spectra of bR obtained from the dark-adapted form which has been chemically bleached and then regenerated with labeled all-tram-[ 14-13C]retinal, while the spectrum of Fig. 2B is taken from a native sample. Both spectra were recorded at room temperature with

MAGIC-ANGLE-SPINNING

DIFFERENCE

A.

Wi3C

bR

B

Natural

abundance bR

C.

Difference

200

100 Chemical

SPECTROSCOPY

0

255

pm

Shift

FIG. 2. MASS NMR spectra of bacteriorhodopsin at vR = 2500 Hz. (A) [ 14-“C]bR, (B) native bR, and (C) the difference between these two. The chemical shifts are relative to external TMS (Si(CH,),). The centerbands of the all-trans- and I3-&retinal of dark-adapted bR are denoted by I and II, respectively.

VR = 2500 Hz. Inspection of Fig. 2A shows that there are two sets of lines in the spectrum which arise from the ‘3C-labeled chromophores and these appear very clearly in the difference spectrum, shown in Fig. 2C. These lines have been previously assigned to the 13-cis and all-trans forms of retinal which occur in dark-adapted bR (16). This difference spectrum was obtained by subtracting the spectrum of the native sample from that of the labeled bR. As is apparent from Fig. 2C, the only peaks which remain are due to the labeled site. The substantial natural abundance background is effectively suppressed except for some minor residual signals around 25 ppm arising from the contributions from the saturated aliphatic carbons in bR and from the Kel-F in the probe. Using the method of Herzfeld and Berger (6) we have calculated the chemical-shift tensors. For the centerband/sideband pattern centered at aI = 110 ppm we obtain ull = 45 ppm, u22 = 107 ppm, and us3 = 179 ppm, whereas for the centerband/sideband pattern centered at UI = 122 ppm, we obtain ull = 50 ppm, u22 = 134 ppm, and u33 = 182 ppm. For both cases the errors in the uii amount to -3 ppm. We have also measured difference spectra at va = 2000 Hz and VR = 3000 Hz, which confirm these results. These values are different from what has been reported previously (16). The difference arises from the greater accuracy available with the difference technique and from the better signal-to-noise ratio which may be achieved by maintaining a constant VRover long data acquisition periods.

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ET AL. REMARKS

We conclude that MASS difference spectra offer important advantages over conventional MASS spectra, since the signal-to-background ratio is substantially enhanced. We have illustrated this point with the example of a “C-labeled protein system. This approach should also be useful for various other MASS experiments in which one is interested in small changes in the spectrum upon variation of externally controllable parameters (e.g., light-induced effects, thermally induced effects, relaxation behavior, and decoupling experiments). Finally, the utility of the spinning speed controller is by no means restricted to the difference technique. In particular, it may be useful for any experiment where either a very precise and well-defined URis needed, as in TOSS experiments (18), or when the spinning speed is required to be constant over long periods of time, as in 2D MASS experiments (19, 20). EXPERIMENTAL

PROCEDURES

The NMR experiments of Fig. 2 were performed with a homebuilt spectrometer and probe. The spinner assemblies were purchased from Doty Scientific, Inc. The 13C frequency of the spectrometer is 79.9 MHz with a 90” 13C pulse length of -6 ps and a 90” ‘H pulse length of -3 ps.. Purple membrane (PM) was obtained from a culture of the JW-3 strain using the purification procedure of Oesterhelt and Stoeckenius (21). The synthesis of 14-i3Clabeled retinal and the procedures for preparing labeled PM are described elsewhere (22). The PM samples were pelleted in a centrifuge before packing them in the rotors. For each spectrum approximately SO,000 transients were accumulated in 5 12 channels with a recycle delay of 2 s and a total spectral width of 50 kHz. After baokground correction, exponential line broadening with 30 Hz, zero-filling to 40% channels, Fourier transformation, and phase adjustment, the spectra of Figs. 2A and 221 were obtained. The subtraction has been done in the form of a fitting procedure, with the phase parameters and an overall scaling factor of the subtracted spectrum as fitting variables. Moreover, we allowed for a small frequency shift of the native spectrum. For this fitting a computer program has been written and has been linked with the CERN computer code MINUIT (23), which provides various kinds of fitting facilities. All calculations were performed on a microVAX. ACKNOWLEDGMENTS We express our sincere gratitude to D. W. Alderman for his kind advice and illuminating discussions. D. J. Ruben is thanked for the loan of his equipment and for many invaluable qgestious concerning the construction of the spinning speed controller. We also thank T. Oas for leading us into the age ofcornpmeraided design, and Ms. A. Lawthers for preparation of the manuscript. This research was suppmtexi by the National Institutes of Health (GM-23403, GM-23289, GM-23316, and RR-O@%), by the Nrethe&& Foundation for Chemical Research (SON), and by the Netherlands Orgsniition for the Advancement of Pure Research (Z.W.O.). H.J.M.d.G. is a recipient of a Z.W.O. Fellowship (SS l-346) and S.O.S. is supported by an NIH Postdoctoral Fellowship (GM-10502).

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REFERENCES SuppI. 1, “Hiph Resolution NMR in Solids: 1. U. HAEBERLEN, “Advances in Magnetic Resonance,” Selective Averaging,” Academic Press, New York, 1976; M. MEHRING, “Principles of High Resolution NMR in Solids,” Springer, Berlin, 1983. 2. R. G. GRIFFIN, W. P. AUE, R. A. HABERKORN, G. S. HARBISON, J. HERZFELD, E. M. MENGER, M. G. MUNOWITZ, E. T. OLEJNICZAK, D. P. RALEIGH, J. E. ROBERTS, D. J. RUBEN, A. SCHMIDT, S. 0. SMITH, AND S. VEGA, “Magic Angle Sample Spinning,” Lecture Notes for the Enrico Fermi School of Physics, July 8-18, 1986, Varenna, Italy, North-Holland, Amsterdam, in press. 3. E. R. ANDREW, A. BRADBURY, AND R. G. EADES, Nature (London) 182, 1659 (1958); I. J. LOWE, Phys. Rev. Lett. 2, 285 (1958). 4. J. SCHAEFER AND E. 0. STEISKAL, J. Am. Chem. Sot. 98, 1031 (1976). 5. A. PINES, M. G. GIBBY, AND J. S. WAUGH, J. Chem. Phys. 59,569 (1973). 6. J. HERZFELD AND A. E. BERGER, J. Chem. Phys. 73,602 1 ( 1980). 7. M. G. MUNOWITZ, R. G. GRIFFIN, G. BODENHAUSEN, AND T.-H. HUANG, J. Am. Chem. Sot. 103, 2529 (1981). 8. (a) L. C. P. J. MOLLEVANGER, A. P. M. KENTGENS, J. A. PARDOEN, J. M. L. COURTIN, W. S. VEEMAN, J. LUGTENBURG, AND W. J. DE GRIP, Eur. J. Biochem. 163,9 (1987); (b) V. BORK, J. SCHAEFER, R. J. AUCHUS, AND D. F. COVEY, “Abstracts 28th ENC, Asilomar, California, April 1987.” 9. J. N. LEE, D. W. ALDERMAN, J. Y. JIN, K. W. ZILM, C. L. MAYNE, R. J. PUGMIRE, AND D. M. GRANT, Rev. Sci. Instrum. 55,516 (1984). 10. Type 1259E5OOOOSV, MKS Instruments, Inc., 6 Shattuck Road, Andover, Massachusetts 018 IO. II. Applied Fiber Optics, Southbridge, Massachusetts; OrieI Corp. P.O. Box 872, Stratford, Connecticut. 12. Type F2516, Edmund Scientific, 101 E. Gloucester Pike, Barrington, New Jersey 08007. 13. D. OE~TERHELT AND W. STOECKENIUS, Nature New Biol. 233, 149 (197 1). 14. R. H. LOZIER, R. A. BOGOMOLNI, AND W. STOECKENIUS, Biophys. J. 15,955 (1975). IS. G. S. HARBISON, S. 0. SMITH, J. A. PARDOEN, J. M. L. COURTIN, J. LUGTENBURG, J. HERZFELD, R. A. MATHIES, AND R. G. GRIPFIN, Biochemistry 24,6955 (1985). 16. G. S. HARBISON, S. 0. SMITH, J. A. PARDOEN, C. WINKEL, J. LUGTENBURG, J. HERZFELD, R. A. MATHIES, AND R. G. GRIFFIN, Proc. Natl. Acad. Sci. USA 81, 1706 (1984). 17. Model 843-5000, C. A. Norgren Co., 5400 S. Delaware St., Littleton, Colorado 80120. 18. W. T. DIXON, J. Magn. Res. 44,220 (1981); J. Chem. Phys. 77, 1800 (1982). 19. M. G. MUNOWITZ AND R. G. GRI~N, J. Chem. Phys. 76,2848 (1982). 20. J. E. ROBERTS, S. VEGA, AND R. G. GRI!=!=IN, J. Am. Chem. Sot. 106,2506 (1984); P. CARAVATTI, L. BRAUNSCHWEILER, AND R. R. ERNST, Chem. Phys. Lett. 100,305 (1983). 21. D. OESTERHELT AND W. STOECKENIUS, in “Methods in Enzymology” (L. Grossman and K. MoI&ve, Eds.), Vol. 31, p. 667, Academic Press, New York, 1973. 22. J. A. PARDOEN, C. WINKEL, P. P. J. MULDER, AND J. LUGTENBURG, Reel. Trav. Chim. Pays-Bas 103, 135 (1984). 23. F. JAMES AND M. ROOS, Mint& computer code, CERN, Geneva, programme D-506.