Efficient Dynamic Nuclear Polarization at 800 MHz/527 GHz with Trityl-Nitroxide Biradicals.

Cross-effect (CE) dynamic nuclear polarization (DNP) is a rapidly developing technique that enhances the signal intensities in magic-angle spinning (MAS) NMR spectra. We report CE DNP experiments at 211, 600, and 800 MHz using a new series of biradical polarizing agents referred to as TEMTriPols, in which a nitroxide (TEMPO) and a trityl radical are chemically tethered. The TEMTriPol molecule with the optimal performance yields a record (1) H NMR signal enhancement of 65 at 800 MHz at a concentration of 10 mM in a glycerol/water solvent matrix. The CE DNP enhancement for the TEMTriPol biradicals does not decrease as the magnetic field is increased in the manner usually observed for bis-nitroxides. Instead, the relatively strong exchange interaction between the trityl and nitroxide moieties determines the magnetic field at which the optimum enhancement is observed.

Restraints on backbone conformations in solid state NMR studies of uniformly labeled proteins from quantitative amide 15N-15N and carbonyl 13C-13C dipolar recoupling data.

Recent structural studies of uniformly (15)N, (13)C-labeled proteins by solid state nuclear magnetic resonance (NMR) rely principally on two sources of structural restraints: (i) restraints on backbone conformation from isotropic (15)N and (13)C chemical shifts, based on empirical correlations between chemical shifts and backbone torsion angles; (ii) restraints on inter-residue proximities from qualitative measurements of internuclear dipole-dipole couplings, detected as the presence or absence of inter-residue crosspeaks in multidimensional spectra. We show that site-specific dipole-dipole couplings among (15)N-labeled backbone amide sites and among (13)C-labeled backbone carbonyl sites can be measured quantitatively in uniformly-labeled proteins, using dipolar recoupling techniques that we call (15)N-BARE and (13)C-BARE (BAckbone REcoupling), and that the resulting data represent a new source of restraints on backbone conformation. (15)N-BARE and (13)C-BARE data can be incorporated into structural modeling calculations as potential energy surfaces, which are derived from comparisons between experimental (15)N and (13)C signal decay curves, extracted from crosspeak intensities in series of two-dimensional spectra, with numerical simulations of the (15)N-BARE and (13)C-BARE measurements. We demonstrate this approach through experiments on microcrystalline, uniformly (15)N, (13)C-labeled protein GB1. Results for GB1 show that (15)N-BARE and (13)C-BARE restraints are complementary to restraints from chemical shifts and inter-residue crosspeaks, improving both the precision and the accuracy of calculated structures.

Segmental polymorphism in a functional amyloid.

Although amyloid fibrils are generally considered to be causative or contributing agents in amyloid diseases, several amyloid fibrils are also believed to have biological functions. Among these are fibrils formed by Pmel17 within melanosomes, which act as a template for melanin deposition. We use solid-state NMR to show that the molecular structures of fibrils formed by the 130-residue pseudo-repeat domain Pmel17:RPT are polymorphic even within the biologically relevant pH range. Thus, biological function in amyloid fibrils does not necessarily imply a unique molecular structure. Solid-state NMR spectra of three Pmel17:RPT polymorphs show that in all cases, only a subset (~30%) of the full amino acid sequence contributes to the immobilized fibril core. Although the repetitive nature of the sequence and incomplete spectral resolution prevent the determination of unique chemical shift assignments from two- and three-dimensional solid-state NMR spectra, we use a Monte Carlo assignment algorithm to identify protein segments that are present in or absent from the fibril core. The results show that the identity of the core-forming segments varies from one polymorph to another, a phenomenon known as segmental polymorphism.

Polarizing agents and mechanisms for high-field dynamic nuclear polarization of frozen dielectric solids.

This article provides an overview of polarizing mechanisms involved in high-frequency dynamic nuclear polarization (DNP) of frozen biological samples at temperatures maintained using liquid nitrogen, compatible with contemporary magic-angle spinning (MAS) nuclear magnetic resonance (NMR). Typical DNP experiments require unpaired electrons that are usually exogenous in samples via paramagnetic doping with polarizing agents. Thus, the resulting nuclear polarization mechanism depends on the electron and nuclear spin interactions induced by the paramagnetic species. The Overhauser Effect (OE) DNP, which relies on time-dependent spin-spin interactions, is excluded from our discussion due the lack of conducting electrons in frozen aqueous solutions containing biological entities. DNP of particular interest to us relies primarily on time-independent, spin-spin interactions for significant electron-nucleus polarization transfer through mechanisms such as the Solid Effect (SE), the Cross Effect (CE) or Thermal Mixing (TM), involving one, two or multiple electron spins, respectively. Derived from monomeric radicals initially used in high-field DNP experiments, bi- or multiple-radical polarizing agents facilitate CE/TM to generate significant NMR signal enhancements in dielectric solids at low temperatures (<100 K). For example, large DNP enhancements (∼300 times at 5 T) from a biologically compatible biradical, 1-(TEMPO-4-oxy)-3-(TEMPO-4-amino)propan-2-ol (TOTAPOL), have enabled high-resolution MAS NMR in sample systems existing in submicron domains or embedded in larger biomolecular complexes. The scope of this review is focused on recently developed DNP polarizing agents for high-field applications and leads up to future developments per the CE DNP mechanism. Because DNP experiments are feasible with a solid-state microwave source when performed at <20K, nuclear polarization using lower microwave power (<100 mW) is possible by forcing a high proportion of biradicals to fulfill the frequency matching condition of CE (two EPR frequencies separated by the NMR frequency) using the strategies involving hetero-radical moieties and/or molecular alignment. In addition, the combination of an excited triplet and a stable radical might provide alternative DNP mechanisms without the microwave requirement.

A general Monte Carlo/simulated annealing algorithm for resonance assignment in NMR of uniformly labeled biopolymers.

We describe a general computational approach to site-specific resonance assignments in multidimensional NMR studies of uniformly (15)N,(13)C-labeled biopolymers, based on a simple Monte Carlo/simulated annealing (MCSA) algorithm contained in the program MCASSIGN2. Input to MCASSIGN2 includes lists of multidimensional signals in the NMR spectra with their possible residue-type assignments (which need not be unique), the biopolymer sequence, and a table that describes the connections that relate one signal list to another. As output, MCASSIGN2 produces a high-scoring sequential assignment of the multidimensional signals, using a score function that rewards good connections (i.e., agreement between relevant sets of chemical shifts in different signal lists) and penalizes bad connections, unassigned signals, and assignment gaps. Examination of a set of high-scoring assignments from a large number of independent runs allows one to determine whether a unique assignment exists for the entire sequence or parts thereof. We demonstrate the MCSA algorithm using two-dimensional (2D) and three-dimensional (3D) solid state NMR spectra of several model protein samples (α-spectrin SH3 domain and protein G/B1 microcrystals, HET-s(218-289) fibrils), obtained with magic-angle spinning and standard polarization transfer techniques. The MCSA algorithm and MCASSIGN2 program can accommodate arbitrary combinations of NMR spectra with arbitrary dimensionality, and can therefore be applied in many areas of solid state and solution NMR.

Quantum mechanical theory of dynamic nuclear polarization in solid dielectrics.

Microwave driven dynamic nuclear polarization (DNP) is a process in which the large polarization present in an electron spin reservoir is transferred to nuclei, thereby enhancing NMR signal intensities. In solid dielectrics there are three mechanisms that mediate this transfer--the solid effect (SE), the cross effect (CE), and thermal mixing (TM). Historically these mechanisms have been discussed theoretically using thermodynamic parameters and average spin interactions. However, the SE and the CE can also be modeled quantum mechanically with a system consisting of a small number of spins and the results provide a foundation for the calculations involving TM. In the case of the SE, a single electron-nuclear spin pair is sufficient to explain the polarization mechanism, while the CE requires participation of two electrons and a nuclear spin, and can be used to understand the improved DNP enhancements observed using biradical polarizing agents. Calculations establish the relations among the electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) frequencies and the microwave irradiation frequency that must be satisfied for polarization transfer via the SE or the CE. In particular, if δ, Δ < ω(0I), where δ and Δ are the homogeneous linewidth and inhomogeneous breadth of the EPR spectrum, respectively, we verify that the SE occurs when ω(M) = ω(0S) ± ω(0I), where ω(M), ω(0S) and ω(0I) are, respectively, the microwave, and the EPR and NMR frequencies. Alternatively, when Δ > ω(0I) > δ, the CE dominates the polarization transfer. This two-electron process is optimized when ω(0S(1))-ω(0S(2)) = ω(0I) and ω(M)~ω(0S(1)) or ω(0S(2)), where ω(0S(1)) and ω(0S(2)) are the EPR Larmor frequencies of the two electrons. Using these matching conditions, we calculate the evolution of the density operator from electron Zeeman order to nuclear Zeeman order for both the SE and the CE. The results provide insights into the influence of the microwave irradiation field, the external magnetic field, and the electron-electron and electron-nuclear interactions on DNP enhancements.

Repeat domains of melanosome matrix protein Pmel17 orthologs form amyloid fibrils at the acidic melanosomal pH.

Most amyloids are pathological, but fragments of Pmel17 form a functional amyloid in vertebrate melanosomes essential for melanin synthesis and deposition. We previously reported that only at the mildly acidic pH (4-5.5) typical of melanosomes, the repeat domain (RPT) of human Pmel17 can form amyloid in vitro. Combined with the known presence of RPT in the melanosome filaments and the requirement of this domain for filament formation, we proposed that RPT may be the core of the amyloid formed in vivo. Although most of Pmel17 is highly conserved across a broad range of vertebrates, the RPT domains vary dramatically, with no apparent homology in some cases. Here, we report that the RPT domains of mouse and zebrafish, as well as a small splice variant of human Pmel17, all form amyloid specifically at mildly acid pH (pH ∼5.0). Protease digestion, mass per unit length measurements, and solid-state NMR experiments suggest that amyloid of the mouse RPT has an in-register parallel ß-sheet architecture with two RPT molecules per layer, similar to amyloid of the Aß peptide. Although there is no sequence conservation between human and zebrafish RPT, amyloid formation at acid pH is conserved.

What can solid state NMR contribute to our understanding of protein folding?

Complete understanding of the folding process that connects a structurally disordered state of a protein to an ordered, biochemically functional state requires detailed characterization of intermediate structural states with high resolution and site specificity. While the intrinsically inhomogeneous and dynamic nature of unfolded and partially folded states limits the efficacy of traditional X-ray diffraction and solution NMR in structural studies, solid state NMR methods applied to frozen solutions can circumvent the complications due to molecular motions and conformational exchange encountered in unfolded and partially folded states. Moreover, solid state NMR methods can provide both qualitative and quantitative structural information at the site-specific level, even in the presence of structural inhomogeneity. This article reviews relevant solid state NMR methods and their initial applications to protein folding studies. Using either chemical denaturation to prepare unfolded states at equilibrium or a rapid freezing apparatus to trap non-equilibrium, transient structural states on a sub-millisecond time scale, recent results demonstrate that solid state NMR can contribute essential information about folding processes that is not available from more familiar biophysical methods.

A Monte Carlo/simulated annealing algorithm for sequential resonance assignment in solid state NMR of uniformly labeled proteins with magic-angle spinning.

We describe a computational approach to sequential resonance assignment in solid state NMR studies of uniformly (15)N,(13)C-labeled proteins with magic-angle spinning. As input, the algorithm uses only the protein sequence and lists of (15)N/(13)C(alpha) crosspeaks from 2D NCACX and NCOCX spectra that include possible residue-type assignments of each crosspeak. Assignment of crosspeaks to specific residues is carried out by a Monte Carlo/simulated annealing algorithm, implemented in the program MC_ASSIGN1. The algorithm tolerates substantial ambiguity in residue-type assignments and coexistence of visible and invisible segments in the protein sequence. We use MC_ASSIGN1 and our own 2D spectra to replicate and extend the sequential assignments for uniformly-labeled HET-s(218-289) fibrils previously determined manually by Siemer et al. (J. Biomol. NMR, 34 (2006) 75-87) from a more extensive set of 2D and 3D spectra. Accurate assignments by MC_ASSIGN1 do not require data that are of exceptionally high quality. Use of MC_ASSIGN1 (and its extensions to other types of 2D and 3D data) is likely to alleviate many of the difficulties and uncertainties associated with manual resonance assignments in solid state NMR studies of uniformly labeled proteins, where spectral resolution and signal-to-noise are often sub-optimal.

Detection of a transient intermediate in a rapid protein folding process by solid-state nuclear magnetic resonance.

We describe the use of solid-state NMR spectroscopy to characterize a partially folded state of the 35-residue helical protein HP35 created by rapid freeze-quenching from a thermally unfolded state on the 10-20 micros time scale. Two-dimensional solid-state (13)C NMR spectra of (13)C-labeled HP35 in frozen glycerol/water solution exhibit two sets of signals, one corresponding to strongly unfolded protein molecules and the other to an ensemble of molecules having native helical secondary structure but incomplete tertiary structure. The NMR data indicate that secondary structure forms within the freeze-quenching time scale but that full folding involves a slower phase of structural annealing. The approximately 5 micros folding time observed in earlier studies of HP35 by time-resolved optical techniques may not represent the time scale for full folding.

Zero-quantum frequency-selective recoupling of homonuclear dipole-dipole interactions in solid state nuclear magnetic resonance.

We describe a method for measuring magnetic dipole-dipole interactions, and hence distances, between pairs of like nuclear spins in a many-spin system under magic-angle spinning (MAS). This method employs a homonuclear dipolar recoupling sequence that creates an average dipole-dipole coupling Hamiltonian under MAS with full zero-quantum symmetry, including both secular and flip-flop terms. Flip-flop terms are then attenuated by inserting rotor-synchronized periods of chemical shift evolution between recoupling blocks, leaving an effective Hamiltonian that contains only secular terms to a good approximation. Couplings between specific pairs of nuclear spins can then be selected with frequency-selective pi pulses. We demonstrate this technique, which we call zero-quantum shift evolution assisted homonuclear recoupling, in a series of one-dimensional and two-dimensional (13)C NMR experiments at 17.6 T and 40.00 kHz MAS frequency on uniformly (13)C-labeled L-threonine powder and on the helix-forming peptide MB(i+4)EK, synthesized with a pair of uniformly (13)C-labeled L-alanine residues. Experimental demonstrations include measurements of distances between (13)C sites that are separated by three bonds, placing quantitative constraints on both sidechain and backbone torsion angles in polypeptides.

Quantitative determination of site-specific conformational distributions in an unfolded protein by solid-state nuclear magnetic resonance.

Solid-state nuclear magnetic resonance (NMR) techniques are used to investigate the structure of the 35-residue villin headpiece subdomain (HP35) in folded, partially denatured, and fully denatured states. Experiments are carried out in frozen glycerol/water solutions, with chemical denaturation by guanidine hydrochloride (GdnHCl). Without GdnHCl, two-dimensional solid-state (13)C NMR spectra of samples prepared with uniform (13)C labeling of selected residues show relatively sharp cross-peaks at chemical shifts that are consistent with the known three-helix bundle structure of folded HP35. At high GdnHCl concentrations, most cross-peaks broaden and shift, qualitatively indicating disruption of the folded structure and development of static conformational disorder in the frozen denatured state. Conformational distributions at one residue in each helical segment are probed quantitatively with three solid-state NMR techniques that provide independent constraints on backbone varphi and psi torsion angles in samples with sequential pairs of carbonyl (13)C labels. Without GdnHCl, the combined data are well fit by alpha-helical conformations. At [GdnHCl]=4.5 M, corresponding to the approximate denaturation midpoint, the combined data are well fit by a combination of alpha-helical and partially extended conformations at each site, but with a site-dependent population ratio. At [GdnHCl]=7.0 M, corresponding to the fully denatured state, the combined data are well fit by a combination of partially extended and polyproline II conformations, again with a site-dependent population ratio. Two entirely different models for conformational distributions lead to nearly the same best-fit distributions, demonstrating the robustness of these conclusions. This work represents the first quantitative investigation of site-specific conformational distributions in partially folded and unfolded states of a protein by solid-state NMR.

High-frequency dynamic nuclear polarization using biradicals: a multifrequency EPR lineshape analysis.

To date, the cross effect (CE) and thermal mixing (TM) mechanisms have consistently provided the largest enhancements in dynamic nuclear polarization (DNP) experiments performed at high magnetic fields. Both involve a three-spin electron-electron-nucleus process whose efficiency depends primarily on two electron-electron interactions--the interelectron distance R and the correct electron paramagnetic resonance (EPR) frequency separation that matches the nuclear Larmor frequency, /omega(e2)-omega(e1)/ = omega(n). Biradicals, for example, two 2,2,6,6-tetramethyl-piperidine-1-oxyls (TEMPOs) tethered with a molecular linker, can in principle constrain both the distance and relative g-tensor orientation between two unpaired electrons, allowing these two spectral parameters to be optimized for the CE and TM. To verify this hypothesis, we synthesized a series of biradicals--bis-TEMPO tethered by n ethylene glycol units (a.k.a. BTnE)--that show an increasing DNP enhancement with a decreasing tether length. Specifically at 90 K and 5 T, the enhancement grew from approximately 40 observed with 10 mM monomeric TEMPO, where the average R approximately 56 A corresponding to electron-electron dipolar coupling constant omega(d)2 pi = 0.3 MHz, to approximately 175 with 5 mM BT2E (10 mM electrons) which has R approximately 13 A with omega(d)2 pi = 24 MHz. In addition, we compared these DNP enhancements with those from three biradicals having shorter and more rigid tethers-bis-TEMPO tethered by oxalyl amide, bis-TEMPO tethered by the urea structure, and 1-(TEMPO-4-oxyl)-3-(TEMPO-4-amino)-propan-2-ol (TOTAPOL) TOTAPOL is of particular interest since it is soluble in aqueous media and compatible with DNP experiments on biological systems such as membrane and amyloid proteins. The interelectron distances and relative g-tensor orientations of all of these biradicals were characterized with an analysis of their 9 and 140 GHz continuous-wave EPR lineshapes. The results show that the largest DNP enhancements are observed with BT2E and TOTAPOL that have shorter tethers and the two TEMPO moieties are oriented so as to satisfy the matching condition for the CE.

Dynamic nuclear polarization at high magnetic fields.

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 (microw) irradiation of the sample. The maximum theoretical enhancement achievable is given by the gyromagnetic ratios (gamma(e)gamma(l)), being approximately 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 (> or =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 microw and probe technology, followed by an overview of DNP applications in biological solids and liquids. Finally, we outline some possible areas for future developments.

High-frequency dynamic nuclear polarization using mixtures of TEMPO and trityl radicals.

In a previous communication [Hu et al., J. Am. Chem. Soc. 126, 10844 (2004)], an approach was demonstrated that improves the efficiency of the cross-effect polarization mechanism employed in high field dynamic nuclear polarization (DNP) experiments. Specifically, it was shown that tethering two TEMPO (2,2,6,6-tetramethylpiperidin-1-oxyl) radicals increases the electron-electron dipole coupling from approximately 1 MHz in solutions of monomeric TEMPO to approximately 25 MHz in a tethered biradical. The larger coupling resulted in an increase in the DNP enhancements by a factor of approximately 3-4, from 45-50 to approximately 165. Here, a second approach to improving the efficiency of the polarization process is described that involves approximately satisfying the matching condition |omega(2e)-omega(1e)|=omega(n), where omega(2e) and omega(1e) are two frequencies in the electron paramagnetic resonance (EPR) spectrum and omega(n) is the Larmor frequency of the nuclear spins being polarized. Specifically, in a mixture of TEMPO and trityl [tris (8-carboxy-2,2,6,6-tetramethyl(-d3)-benzo[1,2d:4,5-d']bis(1,3)dithiol-4-yl) methyl] radicals, the intensity maxima in the EPR spectra of these two species are approximately separated by the (1)H NMR frequency. In this case the frequency difference between the g(yy) value of TEMPO and the narrow pseudo-isotropic g-value of trityl is approximately 224 MHz and the (1)H Larmor frequency is 211 MHz. The optimal magnetic field for DNP using the mixtures was found to coincide with the trityl EPR resonance. At 90 K and 5 T, a mixture of 20 mM TEMPO and 20 mM trityl enhanced the (1)H polarization by a factor of approximately 160, an improvement over the enhancement of approximately 50 with 40 mM TEMPO. The reasons for the improvement are discussed and evidence is presented suggesting that DNP enhancement can be improved further by tethering TEMPO and trityl or two similar radicals.

Spectral Characteristics of a 140-GHz Long-Pulsed Gyrotron.

Gyrotrons operating in the millimeter and submillimeter wavelength ranges are the promising sources for applications that are requiring good spectral characteristics and a wide range of output power. We report the precise measurement results of gyrotron spectra. Experiments were conducted using a 140-GHz long-pulse gyrotron that is developed for the dynamic nuclear polarization/nuclear-magnetic-resonance spectroscopy at the Massachusetts Institute of Technology. Transient downshift of the frequency by 12 MHz with a time constant of 3 s was observed. After reaching equilibrium, the frequency was maintained within 1 ppm for over 20 s. The coefficient of the frequency change with cavity temperature was -2.0 MHz/K, which shows that fine tuning of the gyrotron frequency is plausible by cavity-temperature control. Frequency pulling by the beam current was observed, but it was shown to be masked by the downward shift of the gyrotron frequency with temperature. The linewidth was measured to be much less than 1 MHz at 60 dB relative to the carrier power [in decibels relative to carrier (dBc)] and 4.3 MHz at 75 dBc, which is the largest dynamic range to date for the measurement of gyrotron linewidth to our knowledge.

Dynamic nuclear polarization of amyloidogenic peptide nanocrystals: GNNQQNY, a core segment of the yeast prion protein Sup35p.

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.

TOTAPOL: a biradical polarizing agent for dynamic nuclear polarization experiments in aqueous media.

In a previous publication, we described the use of biradicals, in that case two TEMPO molecules tethered by an ethylene glycol chain of variable length, as polarizing agents for microwave driven dynamic nuclear polarization (DNP) experiments. The use of biradicals in place of monomeric paramagnetic centers such as TEMPO yields enhancements that are a factor of approximately 4 larger (epsilon approximately 175 at 5 T and 90 K) and concurrently the concentration of the polarizing agent is a factor of 4 smaller (10 mM electron spins), reducing the residual electron nuclear dipole broadening. In this paper we describe the synthesis and characterization by EPR and DNP/NMR of an improved polarizing agent 1-(TEMPO-4-oxy)-3-(TEMPO-4-amino)propan-2-ol (TOTAPOL). Under the same experimental conditions and using 2.5 mm magic angle rotors, this new biradical yields larger enhancements (epsilon approximately 290) at lower concentrations (6 mM electron spins) and has the additional important property that it is compatible with experiments in aqueous media, including salt solutions commonly used in the study of proteins and nucleic acids.

In situ temperature jump high-frequency dynamic nuclear polarization experiments: enhanced sensitivity in liquid-state NMR spectroscopy.

We describe an experiment, in situ temperature jump dynamic nuclear polarization (TJ-DNP), that is demonstrated to enhance sensitivity in liquid-state NMR experiments of low-gamma spins--13C, 15N, etc. The approach consists of polarizing a sample at low temperature using high-frequency (140 GHz) microwaves and a biradical polarizing agent and then melting it rapidly with a pulse of 10.6 microm infrared radiation, followed by observation of the NMR signal in the presence of decoupling. In the absence of polarization losses due to relaxation, the enhancement should be epsilon+ = epsilon(T(obs)/T(mu)(wave)), where epsilon+ is the observed enhancement, epsilon is the enhancement obtained at the temperature where the polarization process occurs, and T(mu)(wave) and T(obs) are the polarization and observation temperatures, respectively. In a single experimental cycle, we observe room-temperature enhancements, epsilon(dagger), of 13C signals in the range 120-400 when using a 140 GHz gyrotron microwave source, T(mu)(wave) = 90 K, and T(obs) = 300 K. In addition, we demonstrate that the experiment can be recycled to perform signal averaging that is customary in contemporary NMR spectroscopy. Presently, the experiment is applicable to samples that can be repeatedly frozen and thawed. TJ-DNP could also serve as the initial polarization step in experiments designed for rapid acquisition of multidimensional spectra.

Continuous-wave Submillimeter-wave Gyrotrons.

Recently, dynamic nuclear polarization enhanced nuclear magnetic resonance (DNP/NMR) has emerged as a powerful technique to obtain significant enhancements in spin spectra from biological samples. For DNP in modern NMR systems, a high power continuous-wave source in the submillimeter wavelength range is necessary. Gyrotrons can deliver tens of watts of CW power at submillimeter wavelengths and are well suited for use in DNP/NMR spectrometers. To date, 140 GHz and 250 GHz gyrotrons are being employed in DNP spectrometer experiments at 200 MHz and 380 MHz at MIT. A 460 GHz gyrotron, which has operated with 8 W of CW output power, will soon be installed in a 700 MHz NMR spectrometer. High power radiation with good spectral and spatial resolution from these gyrotrons should provide NMR spectrometers with high signal enhancement through DNP. Also, these tubes operating at submillimeter wavelengths should have important applications in research in physics, chemistry, biology, materials science and medicine.