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Polarization-Enhanced NMR Spectroscopy of Biomolecules in Frozen Solution

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Science  09 May 1997:
Vol. 276, Issue 5314, pp. 930-932
DOI: 10.1126/science.276.5314.930

Abstract

Large dynamic nuclear polarization signal enhancements (up to a factor of 100) were obtained in the solid-state magic-angle spinning nuclear magnetic resonance (NMR) spectra of arginine and the protein T4 lysozyme in frozen glycerol-water solutions with the use of dynamic nuclear polarization. Polarization was transferred from the unpaired electrons of nitroxide free radicals to nuclear spins through microwave irradiation near the electron paramagnetic resonance frequency. This approach may be a generally applicable signal enhancement scheme for the high-resolution solid-state NMR spectroscopy of biomolecules.

Sensitivity often dictates the feasibility of solid-state NMR (1) studies on chemical and biological systems. The small nuclear Zeeman energy splittings result in correspondingly small nuclear spin polarizations at thermal equilibrium. For example, protons exhibit a thermal equilibrium spin polarization of <0.01% at 5 T and 300 K.

We describe here a method that can substantially improve the sensitivity of high-resolution solid-state NMR spectroscopy of biomolecular systems in frozen glycerol-water solutions. We have obtained signal enhancements of up to ∼100 in the 13C spectra of the amino acid arginine and ∼50 in the 15N spectra of the 18.7-kD protein T4-lysozyme, which correspond to a large decrease in signal averaging time or sample size requirements, or both. Alternatively, this approach may permit the routine application of the expanding repertoire of multidimensional homo- and heteronuclear recoupling techniques (2) for performing spectral assignments and determining structural constraints in biomolecular systems such as large soluble proteins, nucleic acids, and membrane proteins.

Dynamic nuclear polarization (DNP) (3) is used to transfer the high spin polarization of unpaired electrons to coupled nuclear spins through microwave irradiation at or near the electron paramagnetic resonance (EPR) frequency. Under optimal conditions, NMR signal intensities can be increased by the ratio of the electronic and nuclear Larmor frequencies, corresponding to factors of ∼660 and ∼2600 for 1H and 13C spins, respectively.

A number of signal enhancement schemes have recently been utilized in NMR spectroscopy, including optical pumping of quantum wells (4), photochemically induced DNP of photosynthetic reaction centers (5), polarization transfer from optically polarized xenon (4, 6) to surface and solution spins, and DNP of diamond, coal, and polymer systems doped with aromatic free radicals (7-9). These approaches have all proven successful for polarizing specific systems, but no method has yet been demonstrated to be generally applicable to macromolecular biological systems. Our DNP enhancement scheme uses a frozen aqueous (60:40 glycerol-water) solution doped with the nitroxide free radical 4-amino-TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy). Glycerol:water is frequently used as a cryoprotectant for protein samples in x-ray crystallography (10), and TEMPO and its analogs are commonly exploited for EPR spin-labeling studies on biological samples (11). Optimum DNP enhancement is achieved at ∼40 mM radical concentration (12).

In the DNP–cross-polarization (CP) pulse sequence (Fig.1), polarization is first transferred from the 4-amino-TEMPO free radical to 1H spins under microwave irradiation. Irradiation on the order of the nuclear spin-lattice relaxation time (T 1) is required for maximal polarization buildup. Proton spin diffusion distributes the enhanced magnetization throughout the solvent and solute during the irradiation period. Finally, a standard CP pulse sequence (13) transfers the high 1H polarization to rare spins (typically13C or 15N) for observation. The magnetic field is chosen to maximize the experimentally determined electron-to-proton polarization transfer efficiency (14).

Figure 1

Diagram of the DNP-CP pulse sequence. Initially, electronic polarization is transferred to proton spins by microwave irradiation for a period on the order of the 1HT 1. The magnetic field is set to maximize the efficiency of polarization transfer. The proton polarization is then transferred to the rare nuclear spins (13C or15N) by a standard cross-polarization sequence. Finally, the 13C or 15N spectrum is acquired while continuous-wave 1H decoupling is performed.

Several mechanisms of electron-proton polarization transfer exist. In these experiments using 40 mM 4-amino-TEMPO in water-glycerol, DNP primarily proceeds through a thermal mixing mechanism (8, 15), in which irradiation off the center of the EPR line coupled with electron-electron cross-relaxation perturbs the electronic dipolar reservoir from thermal equilibrium. The nuclear spins become polarized through their coupling to the electronic dipolar reservoir. This coupling is induced by simultaneous spin flips of two electron spins differing in resonance frequency by the nuclear Zeeman splitting driving a nuclear spin flip (16). Thermal mixing involves irradiation of an allowed EPR transition, in contrast to another DNP mechanism, the solid effect, in which weak, second-order electron-nuclear spin flips are driven (8). Thermal mixing therefore requires less microwave power to achieve maximal enhancement, particularly in high magnetic fields where the probability of the solid effect transition is severely attenuated.

Magic-angle spinning (MAS) (17) is required to obtain high-resolution solid-state NMR spectra. Although MAS techniques are conventionally performed at temperatures above 100 K, the efficiency of the DNP effect increases significantly at low temperature for two major reasons: The leakage of enhanced proton polarization to the lattice is attenuated as T 1 increases, and electron-electron cross-relaxation is more efficient asT 1e increases (18). Our experiments use a DNP-MAS probe that employs helium as the spinning and cooling gas and can achieve spinning speeds up to 5 kHz and temperatures as low as 25 K (19, 20), where the protonT 1 of the 4-amino-TEMPO–water-glycerol system is greater than 30 s, as compared with 10 s or less at temperatures above 100 K.

DNP experiments have typically been limited to low magnetic fields (<1.4 T, corresponding to <40 GHz for EPR and <60 MHz for1H NMR), primarily because of the lack of high-frequency microwave sources that possess sufficient output power and have extended operating lifetimes (21). We have therefore constructed a custom-designed gyrotron (or cyclotron resonance maser) oscillator (22) capable of producing up to 100 W of 140 GHz irradiation under conditions suitable for DNP experiments at 5 T (140 GHz for EPR and 211 MHz for 1H NMR) (23), which affords improved NMR resolution and sensitivity over lower magnetic fields.

Using DNP-CP, we have achieved signal enhancements of up to ∼100 in the MAS spectra of several solutes in the 4-amino-TEMPO–water-glycerol system. As one example, the top spectrum of Fig. 2 shows an enhancement of ∼20 of the13C DNP-CP MAS spectrum of uniformly13C,15N-labeled arginine (30 mg/ml) in frozen solution at 55 K (24). The bottom spectrum was taken under identical conditions with no microwave irradiation.

Figure 2

DNP-CP 13C solid-state MAS (3.0 kHz spin rate) spectra of fully 13C-15N–labeledl-Arg (30 mg/ml Arg and 40 mM 4-amino-TEMPO in 60:40 water-glycerol) at 55 K. The peaks labeled with an asterisk correspond to rotational sidebands and those labeled G correspond to the natural abundance 13C glycerol peaks. Microwave irradiation (139.60 GHz) was performed for 15 s with ∼1 W at the sample. Sixteen acquisitions were averaged with a 1-s recycle delay. The bottom spectrum was recorded under identical conditions with no microwave power. The magnetic field was set to maximize the positive enhancement, which is approximately 20.

T4 lysozyme is a 18.7-kD, 164-residue monomeric protein necessary for the lytic growth of bacteriophage T4. Its structure has been characterized by x-ray diffraction (25) and solution NMR (26), and expression schemes have been developed for the production of isotopically labeled samples (26). Enhancements of approximately 50 were observed in the CP-MAS spectrum of a 1 mM buffered solution of 15N-Ala–labeled T4 lysozyme in TEMPO-water-glycerol at 40 K (Fig. 3). Enhancements of this magnitude may enable the use of newly developed solid-state NMR techniques that are designed for structural studies (2) but currently are difficult to apply to large biological systems.

Figure 3

DNP-CP 15N solid-state MAS (3.2 kHz spin rate) spectra of 15N-Ala–labeled T4 lysozyme [25 mg/ml T4 lysozyme and 40 mM 4-amino-TEMPO in 60% glycerol and 40% buffer (100 mM KCl and 30 mM potassium phosphate)] at 40 K. Microwave irradiation (139.60 GHz) was performed for 20 s with ∼1 W at the sample. Sixty-four acquisitions were averaged with a 15-s recycle delay. The bottom spectrum was recorded under identical conditions with no microwave power. The magnetic field was set to maximize the positive enhancement, which is approximately 50.

The application of this technique to large solutes requires polarization to be delivered from radicals in solution to protons in the solute interior. The most plausible mechanism for this is proton spin diffusion (9). Electronic polarization from the free radical is transferred most efficiently to nearby (primarily solvent) protons, whereas direct transfer to protons within the solute should be extremely slow because of the weak dipolar interaction to the free radical. However, proton spin diffusion is fast compared toT 1, and therefore can potentially deliver enhanced polarization localized around the radical to protons throughout the solvent and solute. Typical spin diffusion constants in protonated solids range from 2 × 10−12 to 6 × 10−12 cm2/s (27), which implies that proton polarization can propagate several hundred angstroms before spin-lattice relaxation causes significant polarization loss (28). This should be sufficiently fast to allow the application of this enhancement technique to biomolecules substantially larger than T4 lysozyme.

There are potential improvements to be made in this enhancement technique. It is possible to implement gyrotron technology to generate microwave radiation in the 200- to 600-GHz range (21) to take advantage of the improved NMR resolution and sensitivity at higher magnetic fields. Also, the overall utility of the DNP technique depends critically on the rate of transfer of polarization from electrons to protons. This transfer scheme requires a time on the order of the nuclear T 1 for maximal polarization transfer. Coherent DNP methods such as electron-nuclear cross-polarization (29) may considerably decrease this transfer time.

  • * Present address: Massachusetts General Hospital–NMR Center, Massachusetts General Hospital, Charlestown, MA 02129, USA.

  • To whom correspondence should be addressed. E-mail: griffin{at}ccnmr.mit.edu

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