Dynamic nuclear polarization (DNP) is a method that permits NMR signal intensities of solids and liquids to be enhanced significantly, and is therefore potentially an important tool in structural and mechanistic studies of biologically relevant molecules. During a DNP experiment, the large polarization of an exogeneous or endogeneous unpaired electron is transferred to the nuclei of interest (I) by microwave (μw) irradiation of the sample. The maximum theoretical enhancement achievable is given by the gyromagnetic ratios (γ e /γ l ), being ∼660 for protons. In the early 1950s, the DNP phenomenon was demonstrated experimentally, and intensively investigated in the following four decades, primarily at low magnetic fields. This review focuses on recent developments in the field of DNP with a special emphasis on work done at high magnetic fields (≥5 T), the regime where contemporary NMR experiments are performed. After a brief historical survey, we present a review of the classical continuous wave (cw) DNP mechanisms-the Overhauser effect, the solid effect, the cross effect, and thermal mixing. A special section is devoted to the theory of coherent polarization transfer mechanisms, since they are potentially more efficient at high fields than classical polarization schemes. The implementation of DNP at high magnetic fields has required the development and improvement of new and existing instrumentation. Therefore, we also review some recent developments in μw and probe technology, followed by an overview of DNP applications in biological solids and liquids. Finally, we outline some possible areas for future developments.

We used solid-state deuterium NMR spectroscopy and an approach involving geometric analysis of labeled alanines (GALA method) to examine the structure and orientation of a designed synthetic hydrophobic, membrane-spanning alpha-helical peptide in phosphatidylcholine (PC) bilayers. The 19-amino-acid peptide consists of an alternating leucine and alanine core, flanked by tryptophans that serve as interfacial anchors: acetyl-GWW(LA)(6)LWWA-ethanolamine (WALP19). A single deuterium-labeled alanine was introduced at different positions within the peptide. Peptides were incorporated in oriented bilayers of dilauroyl- (di-C12:0-), dimyristoyl- (di-C14:0-), or dioleoyl- (di-C18:1(c)-) phosphatidylcholine. The NMR data fit well to a WALP19 orientation characterized by a distinctly nonzero tilt, approximately 4 degrees from the membrane normal, and rapid reorientation about the membrane normal in all three lipids. Although the orientation of WALP19 varies slightly in the different lipids, hydrophobic mismatch does not seem to be the dominant factor causing the tilt. We suggest rather that the peptide itself has an inherently preferred tilted orientation, possibly related to peptide surface characteristics or the disposition of tryptophan indole anchors relative to the lipids, the peptide backbone, and the membrane/water interface. Additionally, the data allow us to define more precisely the local alanine geometry in this membrane-spanning alpha-helix.

The 17-residue N-terminus (httNT) directly flanking the polyQ sequence in huntingtin (htt) N-terminal fragments plays a crucial role in initiating and accelerating the aggregation process that is associated with Huntington’s disease (HD) pathogenesis. Here we report on magic-angle-spinning solid state NMR studies of the amyloid-like aggregates of an htt N-terminal fragment. We find that the polyQ portion of this peptide exists in a rigid, dehydrated amyloid core that is structurally similar to simpler polyQ fibrils and may contain anti-parallel β-sheets. In contrast, the httNT sequence in the aggregates is composed in part of a well-defined helix, which likely also exists in early oligomeric aggregates. Further NMR experiments demonstrate that the N-terminal helical segment displays increased dynamics and water exposure. Given its specific contribution to the initiation, rate and mechanism of fibril formation, the helical nature of httNT and its apparent lack of effect on the polyQ fibril core structure seem surprising. The results provide new details about these disease-associated aggregates, and also provide a clear example of an amino acid sequence that greatly enhances the rate of amyloid formation while itself not taking part in amyloid structure. There is an interesting mechanistic analogy to recent reports pointing out the early stage contributions of transient intermolecular helix-helix interactions in the aggregation behavior of various other amyloid fibrils.

Solid-state NMR methods employing (2)H NMR and geometric analysis of labeled alanines (GALA) were used to study the structure and orientation of the transmembrane alpha-helical peptide acetyl-GWW(LA)(8)LWWA-amide (WALP23) in phosphatidylcholine (PC) bilayers of varying thickness. In all lipids the peptide was found to adopt a transmembrane alpha-helical conformation. A small tilt angle of 4.5 degrees was observed in di-18:1-PC, which has a hydrophobic bilayer thickness that approximately matches the hydrophobic length of the peptide. This tilt angle increased slightly but systematically with increasing positive mismatch to 8.2 degrees in di-C12:0-PC, the shortest lipid used. This small increase in tilt angle is insufficient to significantly change the effective hydrophobic length of the peptide and thereby to compensate for the increasing hydrophobic mismatch, suggesting that tilt of these peptides in a lipid bilayer is energetically unfavorable. The tilt and also the orientation around the peptide axis were found to be very similar to the values previously reported for a shorter WALP19 peptide (GWW(LA)(6)LWWA). As also observed in this previous study, the peptide rotates rapidly around the bilayer normal, but not around its helix axis. Here we show that these properties allow application of the GALA method not only to macroscopically aligned samples but also to randomly oriented samples, which has important practical advantages. A minimum of four labeled alanine residues in the hydrophobic transmembrane sequence was found to be required to obtain accurate tilt values using the GALA method.

Dynamic nuclear polarization (DNP) permits a approximately 10(2)-10(3) enhancement of the nuclear spin polarization and therefore increases sensitivity in nuclear magnetic resonance (NMR) experiments. Here, we demonstrate the efficient transfer of DNP-enhanced (1)H polarization from an aqueous, radical-containing solvent matrix into peptide crystals via (1)H-(1)H spin diffusion across the matrix-crystal interface. The samples consist of nanocrystals of the amyloid-forming peptide GNNQQNY(7-13), derived from the yeast prion protein Sup35p, dispersed in a glycerol-water matrix containing a biradical polarizing agent, TOTAPOL. These crystals have an average width of 100-200 nm, and their known crystal structure suggests that the size of the biradical precludes its penetration into the crystal lattice; therefore, intimate contact of the molecules in the nanocrystal core with the polarizing agent is unlikely. This is supported by the observed differences between the time-dependent growth of the enhanced polarization in the solvent versus the nanocrystals. Nevertheless, DNP-enhanced magic-angle spinning (MAS) spectra recorded at 5 T and 90 K exhibit an average signal enhancement epsilon approximately 120. This is slightly lower than the DNP enhancement of the solvent mixture surrounding the crystals (epsilon approximately 160), and we show that it is consistent with spin diffusion across the solvent-matrix interface. In particular, we correlate the expected DNP enhancement to several properties of the sample, such as crystal size, the nuclear T(1), and the average (1)H-(1)H spin diffusion constant. The enhanced (1)H polarization was subsequently transferred to (13)C and (15)N via cross-polarization, and allowed rapid acquisition of two-dimensional (13)C-(13)C correlation data.