Category Archives: solid-state DNP

Structure of Lipid Nanoparticles Containing siRNA or mRNA by Dynamic Nuclear Polarization-Enhanced NMR Spectroscopy #DNPNMR

Viger-Gravel, J., et al., Structure of Lipid Nanoparticles Containing siRNA or mRNA by Dynamic Nuclear Polarization-Enhanced NMR Spectroscopy. The Journal of Physical Chemistry B, 2018. 122(7): p. 2073-2081.

Here, we show how dynamic nuclear polarization (DNP) NMR spectroscopy experiments permit the atomic level structural characterization of loaded and empty lipid nanoparticles (LNPs). The LNPs used here were synthesized by the microfluidic mixing technique and are composed of ionizable cationic lipid (DLin-MC3-DMA), a phospholipid (distearoylphosphatidylcholine, DSPC), cholesterol, and poly(ethylene glycol) (PEG) (dimyristoyl phosphatidyl ethanolamine (DMPE)–PEG 2000), as well as encapsulated cargoes that are either phosphorothioated siRNA (50 or 100%) or mRNA. We show that LNPs form physically stable complexes with bioactive drug siRNA for a period of 94 days. Relayed DNP experiments are performed to study 1H–1H spin diffusion and to determine the spatial location of the various components of the LNP by studying the average enhancement factors as a function of polarization time. We observe a striking feature of LNPs in the presence and in the absence of encapsulating siRNA or mRNA by comparing our experimental results to numerical spin-diffusion modeling. We observe that LNPs form a layered structure, and we detect that DSPC and DMPE–PEG 2000 lipids form a surface rich layer in the presence (or absence) of the cargoes and that the cholesterol and ionizable cationic lipid are embedded in the core. Furthermore, relayed DNP 31P solid-state NMR experiments allow the location of the cargo encapsulated in the LNPs to be determined. On the basis of the results, we propose a new structural model for the LNPs that features a homogeneous core with a tendency for layering of DSPC and DMPE–PEG at the surface.

Verdazyl-ribose: A new radical for solid-state dynamic nuclear polarization at high magnetic field #DNPNMR

Thurber, K.R., et al., Verdazyl-ribose: A new radical for solid-state dynamic nuclear polarization at high magnetic field. J Magn Reson, 2018. 289: p. 122-131.

Solid-state dynamic nuclear polarization (DNP) using the cross-effect relies on radical pairs whose electron spin resonance (ESR) frequencies differ by the nuclear magnetic resonance (NMR) frequency. We measure the DNP provided by a new water-soluble verdazyl radical, verdazyl-ribose, under both magic-angle spinning (MAS) and static sample conditions at 9.4T, and compare it to a nitroxide radical, 4-hydroxy-TEMPO. We find that verdazyl-ribose is an effective radical for cross-effect DNP, with the best relative results for a non-spinning sample. Under non-spinning conditions, verdazyl-ribose provides roughly 2x larger (13)C cross-polarized (CP) NMR signal than the nitroxide, with similar polarization buildup times, at both 29K and 76K. With MAS at 7kHz and 1.5W microwave power, the verdazyl-ribose does not provide as much DNP as the nitroxide, with the verdazyl providing less NMR signal and a longer polarization buildup time. When the microwave power is decreased to 30mW with 5kHz MAS, the two types of radical are comparable, with the verdazyl-doped sample having a larger NMR signal which compensates for its longer polarization buildup time. We also present electron spin relaxation measurements at Q-band (1.2T) and ESR lineshapes at 1.2 and 9.4T. Most notably, the verdazyl radical has a longer T1e than the nitroxide (9.9ms and 1.3ms, respectively, at 50K and 1.2T). The verdazyl electron spin lineshape is significantly affected by the hyperfine coupling to four (14)N nuclei, even at 9.4T. We also describe 3000-spin calculations to illustrate the DNP potential of possible radical pairs: verdazyl-verdazyl, verdazyl-nitroxide, or nitroxide-nitroxide pairs. These calculations suggest that the verdazyl radical at 9.4T has a narrower linewidth than optimal for cross-effect DNP using verdazyl-verdazyl pairs. Because of the hyperfine coupling contribution to the electron spin linewidth, this implies that DNP using the verdazyl radical would improve at lower magnetic field. Another conclusion from the calculations is that a verdazyl-nitroxide bi-radical would be expected to be slightly better for cross-effect DNP than the nitroxide-nitroxide bi-radicals commonly used now, assuming the same spin-spin coupling constants.

Primary Transfer Step in the Light-Driven Ion Pump Bacteriorhodopsin: An Irreversible U-Turn Revealed by Dynamic Nuclear Polarization-Enhanced Magic Angle Spinning NMR #DNPNMR

Ni, Q.Z., et al., Primary Transfer Step in the Light-Driven Ion Pump Bacteriorhodopsin: An Irreversible U-Turn Revealed by Dynamic Nuclear Polarization-Enhanced Magic Angle Spinning NMR. J. Am. Chem. Soc., 2018. 140(11): p. 4085-4091.

Despite much attention, the path of the highly consequential primary proton transfer in the light-driven ion pump bacteriorhodopsin (bR) remains mysterious. Here we use DNP-enhanced magic angle spinning (MAS) NMR to study critical elements of the active site just before the Schiff base (SB) deprotonates (in the L intermediate), immediately after the SB has deprotonated and Asp85 has become protonated (in the Mo intermediate), and just after the SB has reprotonated and Asp96 has deprotonated (in the N intermediate). An essential feature that made these experiments possible is the 75-fold signal enhancement through DNP. (15)N(SB)-(1)H correlations reveal that the newly deprotonated SB is accepting a hydrogen bond from an alcohol and (13)C-(13)C correlations show that Asp85 draws close to Thr89 before the primary proton transfer. Concurrently, (15)N-(13)C correlations between the SB and Asp85 show that helices C and G draw closer together just prior to the proton transfer and relax thereafter. Together, these results indicate that Thr89 serves to relay the SB proton to Asp85 and that creating this pathway involves rapprochement between the C and G helices as well as chromophore torsion.

DNP-enhanced solid-state NMR spectroscopy of active pharmaceutical ingredients #DNPNMR

Zhao, L., et al., DNP-enhanced solid-state NMR spectroscopy of active pharmaceutical ingredients. Magn. Reson. Chem., 2017. 0(0).

Solid-state NMR spectroscopy has become a valuable tool for the characterization of both pure and formulated active pharmaceutical ingredients (APIs). However, NMR generally suffers from poor sensitivity that often restricts NMR experiments to nuclei with favorable properties, concentrated samples, and acquisition of one-dimensional (1D) NMR spectra. Here, we review how dynamic nuclear polarization (DNP) can be applied to routinely enhance the sensitivity of solid-state NMR experiments by one to two orders of magnitude for both pure and formulated APIs. Sample preparation protocols for relayed DNP experiments and experiments on directly doped APIs are detailed. Numerical spin diffusion models illustrate the dependence of relayed DNP enhancements on the relaxation properties and particle size of the solids and can be used for particle size determination when the other factors are known. We then describe the advanced solid-state NMR experiments that have been enabled by DNP and how they provide unique insight into the molecular and macroscopic structure of APIs. For example, with large sensitivity gains provided by DNP, natural isotopic abundance, (13) C-(13) C double-quantum single-quantum homonuclear correlation NMR spectra of pure APIs can be routinely acquired. DNP also enables solid-state NMR experiments with unreceptive quadrupolar nuclei such as (2) H, (14) N, and (35) Cl that are commonly found in APIs. Applications of DNP-enhanced solid-state NMR spectroscopy for the molecular level characterization of low API load formulations such as commercial tablets and amorphous solid dispersions are described. Future perspectives for DNP-enhanced solid-state NMR experiments on APIs are briefly discussed.

NMR Signal Quenching from Bound Biradical Affinity Reagents in DNP Samples #DNPNMR #NMR

Rogawski, R., et al., NMR Signal Quenching from Bound Biradical Affinity Reagents in DNP Samples. J Phys Chem B, 2017. 121(48): p. 10770-10781.

We characterize the effect of specifically bound biradicals on the NMR spectra of dihydrofolate reductase from E. coli. Dynamic nuclear polarization methods enhance the signal-to-noise of solid state NMR experiments by transferring polarization from unpaired electrons of biradicals to nuclei. There has been recent interest in colocalizing the paramagnetic polarizing agents with the analyte of interest through covalent or noncovalent specific interactions. This experimental approach broadens the scope of dynamic nuclear polarization methods by offering the possibility of selective signal enhancements and the potential to work in a broad range of environments. Paramagnetic compounds can have other effects on the NMR spectroscopy of nearby nuclei, including broadening of nuclear resonances due to the proximity of the paramagnetic agent. Understanding the distance dependence of these interactions is important for the success of the technique. Here we explore paramagnetic signal quenching due to a bound biradical, specifically a biradical-derivatized trimethoprim ligand of E. coli dihydrofolate reductase. Biradical-derivatized trimethoprim has nanomolar affinity for its target, and affords strong and selective signal enhancements in dynamic nuclear polarization experiments. In this work, we show that, although the trimethoprim fragment is well ordered, the biradical (TOTAPOL) moiety is disordered when bound to the protein. The distance dependence in bleaching of NMR signal intensity allows us to detect numerous NMR signals in the protein. We present the possibility that static disorder and electron spin diffusion play roles in this observation, among other contributions. The fact that the majority of signals are observed strengthens the case for the use of high affinity or covalent radicals in dynamic nuclear polarization solid state NMR enhancement.

Solid-State NMR/Dynamic Nuclear Polarization of Polypeptides in Planar Supported Lipid Bilayers #DNPNMR #NMR

Salnikov, E.S., et al., Solid-State NMR/Dynamic Nuclear Polarization of Polypeptides in Planar Supported Lipid Bilayers. J Phys Chem B, 2015. 119(46): p. 14574-83.

Dynamic nuclear polarization has been developed to overcome the limitations of the inherently low signal intensity of NMR spectroscopy. This technique promises to be particularly useful for solid-state NMR spectroscopy where the signals are broadened over a larger frequency range and most investigations rely on recording low gamma nuclei. To extend the range of possible investigations, a triple-resonance flat-coil solid-state NMR probe is presented with microwave irradiation capacities allowing the investigation of static samples at temperatures of 100 K, including supported lipid bilayers. The probe performance allows for two-dimensional separated local field experiments with high-power Lee-Goldberg decoupling and cross-polarization under simultaneous irradiation from a gyrotron microwave generator. Efficient cooling of the sample turned out to be essential for best enhancements and line shape and necessitated the development of a dedicated cooling chamber. Furthermore, a new membrane-anchored biradical is presented, and the geometry of supported membranes was optimized not only for good membrane alignment, handling, stability, and filling factor of the coil but also for heat and microwave dissipation. Enhancement factors of 17-fold were obtained, and a two-dimensional PISEMA spectrum of a transmembrane helical peptide was obtained in less than 2 h.

Chromophore Distortions in Photointermediates of Proteorhodopsin Visualized by Dynamic Nuclear Polarization-Enhanced Solid-State NMR #DNPNMR #NMR #SSNMR

Mehler, M., et al., Chromophore Distortions in Photointermediates of Proteorhodopsin Visualized by Dynamic Nuclear Polarization-Enhanced Solid-State NMR. J. Am. Chem. Soc., 2017. 139(45): p. 16143-16153.

Proteorhodopsin (PR) is the most abundant retinal protein on earth and functions as a light-driven proton pump. Despite extensive efforts, structural data for PR photointermediate states have not been obtained. On the basis of dynamic nuclear polarization (DNP)-enhanced solid-state NMR, we were able to analyze the retinal polyene chain between positions C10 and C15 as well as the Schiff base nitrogen in the ground state in comparison to light-induced, cryotrapped K- and M-states. A high M-state population could be achieved by preventing reprotonation of the Schiff base through a mutation of the primary proton donor (E108Q). Our data reveal unexpected large and alternating (13)C chemical shift changes in the K-state propagating away from the Schiff base along the polyene chain. Furthermore, two different M-states have been observed reflecting the Schiff base reorientation after the deprotonation step. Our study provides novel insight into the photocycle of PR and also demonstrates the power of DNP-enhanced solid-state NMR to bridge the gap between functional and structural data and models.

High-Frequency Dynamic Nuclear Polarization NMR for Solids: Part 1 – An Introduction #DNPNMR

Ha, M. and V.K. Michaelis, High-Frequency Dynamic Nuclear Polarization NMR for Solids: Part 1 – An Introduction, in Modern Magnetic Resonance, G.A. Webb, Editor. 2017, Springer International Publishing: Cham. p. 1-24.

Dynamic nuclear polarization (DNP) NMR spectroscopy, a high-polarization method, is rapidly changing the landscape of what is possible in solid-state nuclear magnetic resonance spectroscopy. To date, there have been over 200 publications discussing high-frequency DNP NMR of solids with more than half being released within the past few years. Below we provide for researchers that may be interested in this high-sensitivity technique an introduction to high-frequency DNP NMR spectroscopy, including instrumentation, mechanisms, polarizing agents, and sample preparation. While there are many applications utilizing high-frequency DNP NMR, Part II will deal with recent advances in method development and applications to biomolecular solids and materials science.

Peptide and Protein Dynamics and Low-Temperature/DNP Magic Angle Spinning NMR #DNPNMR

Ni, Q.Z., et al., Peptide and Protein Dynamics and Low-Temperature/DNP Magic Angle Spinning NMR. J Phys Chem B, 2017. 121(19): p. 4997-5006.

In DNP MAS NMR experiments at approximately 80-110 K, the structurally important -13CH3 and -15NH3+ signals in MAS spectra of biological samples disappear due to the interference of the molecular motions with the 1H decoupling. Here we investigate the effect of these dynamic processes on the NMR line shapes and signal intensities in several typical systems: (1) microcrystalline APG, (2) membrane protein bR, (3) amyloid fibrils PI3-SH3, (4) monomeric alanine-CD3, and (5) the protonated and deuterated dipeptide N-Ac-VL over 78-300 K. In APG, the three-site hopping of the Ala-Cbeta peak disappears completely at 112 K, concomitant with the attenuation of CP signals from other 13C\’s and 15N\’s. Similarly, the 15N signal from Ala-NH3+ disappears at approximately 173 K, concurrent with the attenuation in CP experiments of other 15N\’s as well as 13C\’s. In bR and PI3-SH3, the methyl groups are attenuated at approximately 95 K, while all other 13C\’s remain unaffected. However, both systems exhibit substantial losses of intensity at approximately 243 K. Finally, with spectra of Ala and N-Ac-VL, we show that it is possible to extract site specific dynamic data from the temperature dependence of the intensity losses. Furthermore, 2H labeling can assist with recovering the spectral intensity. Thus, our study provides insight into the dynamic behavior of biological systems over a wide range of temperatures, and serves as a guide to optimizing the sensitivity and resolution of structural data in low temperature DNP MAS NMR spectra.

Efficient assignment and NMR analysis of an intact virus using sequential side-chain correlations and DNP sensitization #DNPNMR

Sergeyev, I.V., et al., Efficient assignment and NMR analysis of an intact virus using sequential side-chain correlations and DNP sensitization. Proc Natl Acad Sci U S A, 2017. 114(20): p. 5171-5176.

An experimental strategy has been developed to increase the efficiency of dynamic nuclear polarization (DNP) in solid-state NMR studies. The method makes assignments simpler, faster, and more reliable via sequential correlations of both side-chain and Calpha resonances. The approach is particularly suited to complex biomolecules and systems with significant chemical-shift degeneracy. It was designed to overcome the spectral congestion and line broadening that occur due to sample freezing at the cryogenic temperatures required for DNP. Nonuniform sampling (NUS) is incorporated to achieve time-efficient collection of multidimensional data. Additionally, fast (25 kHz) magic-angle spinning (MAS) provides optimal sensitivity and resolution. Data collected in <1 wk produced a virtually complete de novo assignment of the coat protein of Pf1 virus. The peak positions and linewidths for samples near 100 K are perturbed relative to those near 273 K. These temperature-induced perturbations are strongly correlated with hydration surfaces.

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