Eugenio Daviso

High Frequency Dynamic Nuclear Polarization

During the three decades 1980-2010, magic angle spinning (MAS) NMR developed into the method of choice to examine many chemical, physical, and biological problems. In particular, a variety of dipolar recoupling methods to measure distances and torsion angles can now constrain molecular structures to high resolution. However, applications are often limited by the low sensitivity of the experiments, due in large part to the necessity of observing spectra of low-γ nuclei such as the I = 1/2 species (13)C or (15)N. The difficulty is still greater when quadrupolar nuclei, such as (17)O or (27)Al, are involved. This problem has stimulated efforts to increase the sensitivity of MAS experiments. A particularly powerful approach is dynamic nuclear polarization (DNP) which takes advantage of the higher equilibrium polarization of electrons (which conventionally manifests in the great sensitivity advantage of EPR over NMR). In DNP, the sample is doped with a stable paramagnetic polarizing agent and irradiated with microwaves to transfer the high polarization in the electron spin reservoir to the nuclei of interest. The idea was first explored by Overhauser and Slichter in 1953. However, these experiments were carried out on static samples, at magnetic fields that are low by current standards. To be implemented in contemporary MAS NMR experiments, DNP requires microwave sources operating in the subterahertz regime, roughly 150-660 GHz, and cryogenic MAS probes. In addition, improvements were required in the polarizing agents, because the high concentrations of conventional radicals that are required to produce significant enhancements compromise spectral resolution. In the last two decades, scientific and technical advances have addressed these problems and brought DNP to the point where it is achieving wide applicability. These advances include the development of high frequency gyrotron microwave sources operating in the subterahertz frequency range. In addition, low temperature MAS probes were developed that permit in situ microwave irradiation of the samples. And, finally, biradical polarizing agents were developed that increased the efficiency of DNP experiments by factors of ∼4 at considerably lower paramagnet concentrations. Collectively, these developments have made it possible to apply DNP on a routine basis to a number of different scientific endeavors, most prominently in the biological and material sciences. This Account reviews these developments, including the primary mechanisms used to transfer polarization in high frequency DNP, and the current choice of microwave sources and biradical polarizing agents. In addition, we illustrate the utility of the technique with a description of applications to membrane and amyloid proteins that emphasizes the unique structural information that is available in these two cases.

Dynamic nuclear polarization at 700 MHz/460 GHz

We describe the design and implementation of the instrumentation required to perform DNP-NMR at higher field strengths than previously demonstrated, and report the first magic-angle spinning (MAS) DNP-NMR experiments performed at (1)H/e(-) frequencies of 700 MHz/460 GHz. The extension of DNP-NMR to 16.4 T has required the development of probe technology, cryogenics, gyrotrons, and microwave transmission lines. The probe contains a 460 GHz microwave channel, with corrugated waveguide, tapers, and miter-bends that couple microwave power to the sample. Experimental efficiency is increased by a cryogenic exchange system for 3.2 mm rotors within the 89 mm bore. Sample temperatures ≤85 K, resulting in improved DNP enhancements, are achieved by a novel heat exchanger design, stainless steel and brass vacuum jacketed transfer lines, and a bronze probe dewar. In addition, the heat exchanger is preceded with a nitrogen drying and generation system in series with a pre-cooling refrigerator. This reduces liquid nitrogen usage from >700 l per day to <200 l per day and allows for continuous (>7 days) cryogenic spinning without detrimental frost or ice formation. Initial enhancements, ε=-40, and a strong microwave power dependence suggests the possibility for considerable improvement. Finally, two-dimensional spectra of a model system demonstrate that the higher field provides excellent resolution, even in a glassy, cryoprotecting matrix.

An Amyloid Organelle, Solid-state NMR Evidence for Cross-β Assembly of Gas Vesicles

Background: The gas vesicles of aquatic microorganisms are hollow proteinaceous shells with remarkable physical properties that enable them to function as floatation organelles. Results: The gas vesicle subunits associate in a cross-β arrangement. Conclusion: The gas vesicle wall constitutes a functional amyloid. Significance: This new category of functional amyloid broadens our understanding of the diverse roles of the amyloid fold. Functional amyloids have been identified in a wide range of organisms, taking on a variety of biological roles and being controlled by remarkable mechanisms of directed assembly. Here, we report that amyloid fibrils constitute the ribs of the buoyancy organelles of Anabaena flos-aquae. The walls of these gas-filled vesicles are known to comprise a single protein, GvpA, arranged in a low pitch helix. However, the tertiary and quaternary structures have been elusive. Using solid-state NMR correlation spectroscopy we find detailed evidence for an extended cross-β structure. This amyloid assembly helps to account for the strength and amphiphilic properties of the vesicle wall. Buoyancy organelles thus dramatically extend the scope of known functional amyloids.

Molecular structure of humin and melanoidin via solid state NMR.

Sugar-derived humins and melanoidins figure significantly in food chemistry, agricultural chemistry, biochemistry, and prebiotic chemistry. Despite wide interest and significant experimental attention, the amorphous and insoluble nature of the polymers has made them resistant to conventional structural characterization. Here we make use of solid-state NMR methods, including selective (13)C substitution, (1)H-dephasing, and double quantum filtration. The spectra, and their interpretation, are simplified by relying exclusively on hydronium for catalysis. The results for polymers derived from ribose, deoxyribose, and fructose indicate diverse pathways to furans, suggest a simple route to pyrroles in the presence of amines, and reveal a heterogeneous network-type polymer in which sugar molecules cross-link the heterocycles.

Efficient resonance assignment of proteins in MAS NMR by simultaneous intra- and inter-residue 3D correlation spectroscopy.

Resonance assignment is the first step in NMR structure determination. For magic angle spinning NMR, this is typically achieved with a set of heteronuclear correlation experiments (NCaCX, NCOCX, CONCa) that utilize SPECIFIC-CP 15N–13C transfers. However, the SPECIFIC-CP transfer efficiency is often compromised by molecular dynamics and probe performance. Here we show that one-bond ZF-TEDOR 15N–13C transfers provide simultaneous NCO and NCa correlations with at least as much sensitivity as SPECIFIC-CP for some non-crystalline samples. Furthermore, a 3D ZF-TEDOR-CC experiment provides heteronuclear sidechain correlations and robustness with respect to proton decoupling and radiofrequency power instabilities. We demonstrate transfer efficiencies and connectivities by application of 3D ZF-TEDOR-DARR to a model microcrystalline protein, GB1, and a less ideal system, GvpA in intact gas vesicles.

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

In DNP MAS NMR experiments at ∼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-Cβ peak disappears completely at 112 K, concomitant with the attenuation of CP signals from other 13Cs and 15Ns. Similarly, the 15N signal from Ala-NH3+ disappears at ∼173 K, concurrent with the attenuation in CP experiments of other 15Ns as well as 13Cs. In bR and PI3-SH3, the methyl groups are attenuated at ∼95 K, while all other 13Cs remain unaffected. However, both systems exhibit substantial losses of intensity at ∼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.

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

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. 15N(SB)-1H correlations reveal that the newly deprotonated SB is accepting a hydrogen bond from an alcohol and 13C-13C correlations show that Asp85 draws close to Thr89 before the primary proton transfer. Concurrently, 15N-13C 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.