Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register

The yeast prion protein Sup35NM is a self-propagating amyloid. Despite intense study, there is no consensus on the organization of monomers within Sup35NM fibrils. Some studies point to a â-helical arrangement, whereas others suggest a parallel inregister organization. Intermolecular contacts are often determined by experiments that probe long-range heteronuclear contacts for fibrils templated from a 1:1 mixture of 13 C- and 15 N-labeled monomers. However, for Sup35NM, like many large proteins, chemical shift degeneracy limits the usefulness of this approach. Segmental and specific isotopic labeling reduce degeneracy, but experiments to measure long-range interactions are often too insensitive. To limit degeneracy and increase experimental sensitivity, we combined specific and segmental isotopic labeling schemes with dynamic nuclear polarization (DNP) NMR. Using this combination, we examined an amyloid form of Sup35NM that does not have a parallel in-register structure. The combination of a small number of specific labels with DNP NMR enables determination of architectural information about polymeric protein systems. Keyword: [PSI+] prion; solid-state NMR; amyloid; Sup35; dynamic nuclear polarizationNational Institutes of Health (U.S.) (Grants GM-025874)National Institutes of Health (U.S.) (Grants EB-003151)National Institutes of Health (U.S.) (Grants EB-002804)National Institutes of Health (U.S.) (Grants EB-002026

Dynamic Nuclear Polarization-Enhanced Solid-State NMR Spectroscopy of GNNQQNY Nanocrystals and Amyloid Fibrils

Dynamic nuclear polarization (DNP) utilizes the inherently larger polarization of electrons to enhance the sensitivity of conventional solid-state NMR experiments at low temperature. Recent advances in instrumentation development and sample preparation have transformed this field and have opened up new opportunities for its application to biological systems. Here, we present DNP-enhanced [superscript 13]C–[superscript 13]C and [superscript 15]N–[superscript 13]C correlation experiments on GNNQQNY nanocrystals and amyloid fibrils acquired at 9.4 T and 100 K and demonstrate that DNP can be used to obtain assignments and site-specific structural information very efficiently. We investigate the influence of temperature on the resolution, molecular conformation, structural integrity and dynamics in these two systems. In addition, we assess the low-temperature performance of two commonly used solid-state NMR experiments, proton-driven spin diffusion (PDSD) and transferred echo double resonance (TEDOR), and discuss their potential as tools for measurement of structurally relevant distances at low temperature in combination with DNP.National Institutes of Health (U.S.) (Grant EB002804)National Institutes of Health (U.S.) (Grant EB003151)National Institutes of Health (U.S.) (Grant EB002026

Solid-state NMR enhanced by dynamic nuclear polarization as a novel tool for ribosome structural biology

The impact of Nuclear Magnetic Resonance (NMR) on studies of large macromolecular complexes hinges on improvements in sensitivity and resolution. Dynamic nuclear polarization (DNP) in the solid state can offer improved sensitivity, provided sample preparation is optimized to preserve spectral resolution. For a few nanomoles of intact ribosomes and an 800 kDa ribosomal complex we demonstrate that the combination of DNP and magic-angle spinning NMR (MAS-NMR) allows one to overcome current sensitivity limitations so that homo- and heteronuclear C-13 and N-15 NMR correlation spectra can be recorded. Ribosome particles, directly pelleted and frozen into an NMR rotor, yield DNP signal enhancements on the order of similar to 25-fold and spectra that exhibit narrow linewidths, suitable for obtaining site-specific information. We anticipate that the same approach is applicable to other high molecular weight complexes

Extending Timescales and Narrowing Linewidths in NMR

Among the different fields of research in nuclear magnetic resonance (NMR) which are currently investigated in the Laboratory of Biomolecular Magnetic Resonance (LRMB), two subjects that are closely related to each other are presented in this article. On the one hand, we show how to populate long-lived states (LLS) that have long lifetimes T_LLS which allow one to go beyond the usual limits imposed by the longitudinal relaxation time T_1. This makes it possible to extend NMR experiments to longer time-scales. As an application, we demonstrate the extension of the timescale of diffusion measurements by NMR spectroscopy. On the other hand, we review our work on long-lived coherences (LLC), a particular type of coherence between two spin states that oscillates with the frequency of the scalar coupling constant J_IS and decays with a time constant T_LLC. Again, this time constant T_LLC can be much longer than the transverse relaxation time T_2. By extending the coherence lifetimes, we can narrow the linewidths to an unprecedented extent. J-couplings and residual dipolar couplings (RDCs) in weakly-oriented phases can be measured with the highest precision

Dynamic Nuclear Polarization NMR Spectroscopy Allows High-Throughput Characterization of Microporous Organic Polymers

Dynamic nuclear polarization (DNP) solid-state NMR was used to obtain natural abundance 13C and 15N CP MAS NMR spectra of microporous organic polymers with excellent signal-to-noise ratio, allowing for unprecedented details in the molecular structure to be determined for these complex polymer networks. Sensitivity enhancements larger than 10 were obtained with bis-nitroxide radical at 14.1 T and low temperature (∼105 K). This DNP MAS NMR approach allows efficient, high-throughput characterization of libraries of porous polymers prepared by combinatorial chemistry methods

Broadband excitation and indirect detection of nitrogen-14 in rotating solids using Delays Alternating with Nutation (DANTE)

A train of short rotor-synchronized pulses in the manner of Delays Alternating with Nutations for Tailored Excitation (DANTE) applied to nitrogen-14 nuclei (I = 1) in samples spinning at the magic angle at high frequencies (typically nu(rot) = 62.5 kHz so that tau(rot) = 16 mu s) allows one to achieve uniform excitation of a great number of spinning sidebands that arise from large first-order quadrupole interactions, as occur for aromatic nitrogen-14 nuclei in histidine. With routine rf amplitudes omega(1)(N-14)/(2 pi) = 60 kHz and very short pulses of a typical duration 0.5 < tau(p) < 2 mu s, efficient excitation can be achieved with 13 rotor-synchronized pulses in 13 tau(rot) = 208 mu s. Alternatively, with 'overtone' DANTE sequences using 2, 4, or 8 pulses per rotor period one can achieve efficient broadband excitation in fewer rotor periods, typically 2-4 tau(rot). These principles can be combined with the indirect detection of N-14 nuclei via spy nuclei with S = 1/2 such as H-1 or C-13 in the manner of Dipolar Heteronuclear Multiple-Quantum Correlation (D-HMQC). (C) 2011 Elsevier Inc. All rights reserved

A spinning thermometer to monitor microwave heating and glass transitions in dynamic nuclear polarization

As previously demonstrated by Thurber and Tycko, the peak position of Br-79 in potassium bromide (KBr) allows one to determine the temperature of a spinning sample. We propose to adapt the original design by using a compact KBr tablet placed at the bottom of the magic angle spinning rotor, separated from the sample under investigation by a thin disk made of polytetrafluoroethylene (or 'Teflon'(R)). This design allows spinning the sample up to at least 16 kHz. The KBr tablet can remain in the rotor when changing the sample under investigation. Calibration in the range of 98 < T < 320 K has been carried out in a static rotor by inserting a platinum thermometer. The accuracy is better than +/- 0.9 K, even in the presence of microwave irradiation. Irradiation with 5W microwaves at 263 GHz leads to a small temperature increase of 3.6 +/- 1.4 K in either static or spinning samples. The dynamic nuclear polarization enhancement decreases with increasing temperature, in particular when a frozen glassy sample undergoes a glass transition. Copyright (C) 2011 John Wiley & Sons, Ltd

Dynamic Nuclear Polarization NMR Enables the Analysis of Sn-Beta Zeolite Prepared with Natural Abundance [superscript 119]Sn Precursors

The catalytic activity of tin-containing zeolites, such as Sn-Beta, is critically dependent on the successful incorporation of the tin metal center into the zeolite framework. However, synchrotron-based techniques or solid-state nuclear magnetic resonance (ssNMR) of samples enriched with [superscript 119]Sn isotopes are the only reliable methods to verify framework incorporation. This work demonstrates, for the first time, the use of dynamic nuclear polarization (DNP) NMR for characterizing zeolites containing ~2 wt % of natural abundance Sn without the need for [superscript 119]Sn isotopic enrichment. The biradicals TOTAPOL, bTbK, bCTbK, and SPIROPOL functioned effectively as polarizing sources, and the solvent enabled proper transfer of spin polarization from the radical’s unpaired electrons to the target nuclei. Using bCTbK led to an enhancement (ε) of 75, allowing the characterization of natural-abundance [superscript 119]Sn-Beta with excellent signal-to-noise ratios in <24 h. Without DNP, no [superscript 119]Sn resonances were detected after 10 days of continuous analysis.United States. Dept. of Energy. Office of Basic Energy Sciences (Chemical Sciences, Geosciences and Biosciences Dvision DE-FG02-12ER16352)National Institutes of Health (U.S.) (Grant EB-002804)National Institutes of Health (U.S.) (Grant EB-001960)National Institutes of Health (U.S.) (Grant EB-002026)Natural Sciences and Engineering Research Council of Canada (Postdoctoral Fellowship

Sensitivity-enhanced solid-state NMR detection of expansin\u27s target in plant cell walls

Structure determination of protein binding to noncrystalline macromolecular assemblies such as plant cell walls (CWs) poses a significant structural biology challenge. CWs are loosened during growth by expansin proteins, which weaken the noncovalent network formed by cellulose, hemicellulose, and pectins, but the CW target of expansins has remained elusive because of the minute amount of the protein required for activity and the complex nature of the CW. Using solid-state NMR spectroscopy, combined with sensitivity-enhancing dynamic nuclear polarization (DNP) and differential isotopic labeling of expansin and polysaccharides, we have now determined the functional binding target of expansin in the Arabidopsis thaliana CW. By transferring the electron polarization of a biradical dopant to the nuclei, DNP allowed selective detection of 13C spin diffusion from trace concentrations of 13C, 15N-labeled expansin in the CW to nearby polysaccharides. From the spin diffusion data of wild-type and mutant expansins, we conclude that to loosen the CW, expansin binds highly specific cellulose domains enriched in xyloglucan, whereas more abundant binding to pectins is unrelated to activity. Molecular dynamics simulations indicate short 13C-13C distances of 4-6 A between a hydrophobic surface of the cellulose microfibril and an aromatic motif on the expansin surface, consistent with the observed NMR signals. DNP-enhanced 2D 13C correlation spectra further reveal that the expansin-bound cellulose has altered conformation and is enriched in xyloglucan, thus providing unique insight into the mechanism of CW loosening. DNP-enhanced NMR provides a powerful, generalizable approach for investigating protein binding to complex macromolecular targets