Sensitivity-enhanced NMR of biological solids: dynamic nuclear polarization of Y21M fd bacteriophage and purple membrane.

Solid-state NMR (SSNMR) is established as a powerful tool for extracting structural parameters from biological and chemical systems.1-3 However, the inherent low sensitivity of NMR often limits the scope of structural studies. With dynamic nuclear polarization (DNP) it is possible to address this issue since signal intensities can be enhanced by 2 to 3 orders of magnitude. This gain in sensitivity is accomplished by transferring polarization from electrons in exogenous paramagnetic molecules to nuclear spins via microwave irradiation at or near the EPR Larmor frequency.4 The size of the DNP signal enhancement is dependent on a number of factors including the nuclear spin lattice relaxation time, T1, which is maximized by performing experiments at low temperatures. Systems with an abundance of methyl groups, such as virus particles and membrane proteins, often have short T1’s even at low temperatures and could be particularly challenging candidates for DNP experiments. However, these are also some of the most interesting cases for SSNMR structural studies since they often cannot be examined with either solution NMR or X-ray diffraction. In this communication we demonstrate that it is possible to efficiently polarize archetypal examples of each of the systems mentioned above-namely the viral particle fd and the purple membrane containing bacteriorhodopsin (bR) and its accompanying lipids. Furthermore, by comparing the DNP signal enhancements in the 15N and 31P spectra of fd bacteriophage, we show that 1H spin diffusion evenly distributes the enhanced polarization throughout a large macromolecular assembly. These results suggest that DNP may be a generally applicable approach for sensitivity enhancement in SSNMR experiments. We have been developing DNP techniques for studies at high magnetic fields [initially at 5 T and more recently at 9 T (140 and 250 GHz EPR and 211 and 380 MHz 1H NMR frequencies)]. To date, our most successful polarization transfer experiments are based on thermal mixing5,6 with the nitroxide radical 4-aminoTEMPO doped into water/glycerol. The experiment is implemented as illustrated in Figure 1 and consists of microwave irradiation and cross polarization from 1H to a low-γ nucleus. We have achieved 13C signal enhancements of ∼400 in H2O/ glycerol mixtures at 13 K using a microwave cavity7 and ∼50 in the 15N magic angle spinning (MAS) spectrum of T4 lysozyme at 50 K,8 both at 5 T. Filamentous fd bacteriophages are long rod-shaped viruses comprising a 6408 nucleotide single-stranded DNA genome surrounded by 2700 copies of the R-helical pVIII (major coat) protein. The dimensions of a single virion are 8000 × 65 A.9 Due to the shape anisotropy and the alignment of the coat proteins approximately parallel to the virion long axis, the phage particles orient in a magnetic field, enabling high-resolution static SSNMR experiments.10,11 The Y21M mutation of the coat protein decreases the conformational heterogeneity of the coat protein assembly and increases the NMR spectral resolution for oriented samples.12 Since the fd virus is ∼90 wt % protein and 10 wt % DNA, the 15N NMR spectrum is dominated by signals from the coat protein backbone. In contrast, the 31P spectrum is due exclusively to the encapsulated DNA.10 Powder patterns for uniformly 15N-labeled Y21M fd bacteriophage in TEMPO/water/glycerol are shown in Figure 2 b and c at 13 K and 5 T with and without DNP, respectively. Since the fd bacteriophage orients in a magnetic field, the sample was frozen prior to insertion into the precooled probe to obtain a powder pattern. The resulting signal is fit with Ω ) 168 ppm and κ ) -0.75, representative of an amide backbone (Figure 2 a). The narrow peak on the upfield edge of the experimental powder pattern arises from the amino groups of the five lysine residues and the N terminus. The signal enhancement due to DNP is 26, representing a factor of approximately 675 reduction in signal averaging time. An additional factor of 20 in signal intensity is gained from the increased Bolztmann polarization by performing NMR experiments at 15 K versus room temperature. The 1H T1 ) 15 s at 13 K. The high number of methylated amino side chains on the coat protein contribute to this short T1; specifically, the 50-residue major coat protein of Y21M fd contains 10 Ala, 4 Ile, 2 Leu, 2 Met, 3 Thr, and 4 Val residues.9 Despite the short 1H T1 at low temperatures, a substantial signal enhancement is observed with DNP. To use DNP as a sensitivity enhancement tool for structural studies it is desirable to have uniform signal enhancements throughout the sample. In a typical DNP experiment, the bulk protons are polarized followed by cross-polarization (CP) from 1H’s to a low-γ nucleus prior to detection (Figure 1). Since 1H’s are highly abundant, we have argued that rapid spin-diffusion distributes the enhanced polarization throughout the solvent and solute.8 Thus, with the fd bacteriophage 1H spin diffusion should mediate the diffusion of polarization from the solvent to the encapsulated DNA, through the 20 A layer of coat proteins. Figure 2 e and f show 31P powder patterns with and without DNP acquired immediately after the 15N experiment (with no change in temperature) and a similar signal enhancement is observed, ∼26. Detection of equal DNP enhancements on the coat protein and DNA core confirms that 1H spin diffusion is an efficient mechanism for uniformly distributing enhanced polarization * Address correspondence to this author. ‡ Department of Chemistry, MIT. † Current address: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093. § Department of Chemistry, Brandeis University. (1) Opella, S. J. Nat. Struct. Biol. 1997, 4, 845-848. (2) Griffin, R. G. Nat. Struct. Biol. 1998, 5, 508-512. (3) van Beek, J. D.; Beaulieu, L.; Schafer, H.; Demura, M.; Asakura, T.; Meier, B. H. Nature 2000, 405, 1077-1079. (4) Abragam, A. The Principles of Nuclear Magnetism; Clarendon: Oxford, England, 1961. (5) Wenckebach, W. T.; Swanenburg, T. J. B.; Poulis, N. J. Phys. Rep. 1974, 14, 181-255. (6) Duijvestijn, M. J.; Wind, R. A.; Smidt, J. Physica B 1986, 138, 147170. (7) Weis, V.; Bennati, M.; Rosay, M.; Bryant, J. A.; Griffin, R. G. J. Magn. Reson. 1999, 140, 293-299. (8) Hall, D. A.; Maus, D. C.; Gerfen, G. J.; Inati, S. J.; Becerra, L. R.; Dahlquist, F. W.; Griffin, R. G. Science 1997, 276, 930-2. (9) Marvin, D. A.; Hale, R. D.; Nave, C. J. Mol. Biol. 1994, 235, 260286. (10) Cross, T. A.; Tsang, P.; Opella, S. J. Biochemistry 1983, 22, 721726. (11) Cross, T. A.; Frey, M. H.; Opella, S. J. J. Am. Chem. Soc. 1983, 105, 7471-7473. (12) Tan, W. M.; Jelinek, R.; Opella, S. J.; Malik, P.; Terry, T. D.; Perham, R. N. J. Mol. Biol. 1999, 286, 787-796. 1010 J. Am. Chem. Soc. 2001, 123, 1010-1011