Brute-Force Hyperpolarization for NMR and MRI

Hyperpolarization (HP) of nuclear spins is critical for ultrasensitive nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI). We demonstrate an approach for >1500-fold enhancement of key small-molecule metabolites: 1-¹³C-pyruvic acid, 1-¹³C-sodium lactate, and 1-¹³C-acetic acid. The ¹³C solution NMR signal of pyruvic acid was enhanced 1600-fold at <i>B</i> = 1 T and 40 °C by pre-polarizing at 14 T and ∼2.3 K. This “brute-force” approach uses only field and temperature to generate HP. The noted 1 T observation field is appropriate for benchtop NMR and near the typical 1.5 T of MRI, whereas high-field observation scales enhancement as 1/<i>B</i>. Our brute-force process ejects the frozen, solid sample from the low-<i>T</i>, high-<i>B</i> polarizer, passing it through low field (<i>B</i> < 100 G) to facilitate “thermal mixing”. That equilibrates ¹H and ¹³C in hundreds of milliseconds, providing ¹³C HP from ¹H Boltzmann polarization attained at high <i>B</i>/<i>T</i>. The ejected sample arrives at a room-temperature, permanent magnet array, where rapid dissolution with 40 °C water yields HP solute. Transfer to a 1 T NMR system yields ¹³C signals with enhancements at 80% of ideal for noted polarizing conditions. High-resolution NMR of the same product at 9.4 T had consistent enhancement plus resolution of ¹³C shifts and <i>J</i>-couplings for pyruvic acid and its hydrate. Comparable HP was achieved with frozen aqueous lactate, plus notable enhancement of acetic acid, demonstrating broader applicability for small-molecule NMR and metabolic MRI. Brute-force avoids co-solvated free-radicals and microwaves that are essential to competing methods. Here, unadulterated samples obviate concerns about downstream purity and also exhibit slow solid-state spin relaxation, favorable for transporting HP samples

Dynamic Nuclear Polarization Enhanced Natural Abundance ¹⁷O Spectroscopy

We show that natural abundance oxygen-17 NMR of solids could be obtained in minutes at a moderate magnetic field strength by using dynamic nuclear polarization (DNP). Electron spin polarization could be transferred either directly to ¹⁷O spins or indirectly via ¹H spins in inorganic oxides and hydroxides using an oxygen-free solution containing a biradical polarization agent (bTbK). The results open up a powerful method for rapidly acquiring high signal-to-noise ratio solid-state NMR spectra of ¹⁷O nuclear spins and to probe sites on or near the surface, without the need for isotope labeling

Dynamic Nuclear Polarization Study of Inhibitor Binding to the M2_18–60 Proton Transporter from Influenza A

We demonstrate the use of dynamic nuclear polarization (DNP) to elucidate ligand binding to a membrane protein using dipolar recoupling magic angle spinning (MAS) NMR. In particular, we detect drug binding in the proton transporter M2_18–60 from influenza A using recoupling experiments at room temperature and with cryogenic DNP. The results indicate that the pore binding site of rimantadine is correlated with previously reported widespread chemical shift changes, suggesting functional binding in the pore. Futhermore, the ¹⁵N-labeled ammonium of rimantadine was observed near A30 ¹³Cβ and G34 ¹³Cα, suggesting a possible hydrogen bond to A30 carbonyl. Cryogenic DNP was required to observe the weaker external binding site(s) in a ZF-TEDOR spectrum. This approach is generally applicable, particularly for weakly bound ligands, in which case the application of MAS NMR dipolar recoupling requires the low temperatures to quench dynamic exchange processes. For the fully protonated samples investigated, we observed DNP signal enhancements of ∼10 at 400 MHz using only 4–6 mM of the polarizing agent TOTAPOL. At 600 MHz and with DNP, we measured a distance between the drug and the protein to a precision of 0.2 Å

Atomic Resolution Structure of Monomorphic Aβ₄₂ Amyloid Fibrils

Amyloid-β (Aβ) is a 39–42 residue protein produced by the cleavage of the amyloid precursor protein (APP), which subsequently aggregates to form cross-β amyloid fibrils that are a hallmark of Alzheimer’s disease (AD). The most prominent forms of Aβ are Aβ_1–40 and Aβ_1–42, which differ by two amino acids (I and A) at the C-terminus. However, Aβ₄₂ is more neurotoxic and essential to the etiology of AD. Here, we present an atomic resolution structure of a monomorphic form of Aβ_M01–42 amyloid fibrils derived from over 500 ¹³C–¹³C, ¹³C–¹⁵N distance and backbone angle structural constraints obtained from high field magic angle spinning NMR spectra. The structure (PDB ID: 5KK3) shows that the fibril core consists of a dimer of Aβ₄₂ molecules, each containing four β-strands in a S-shaped amyloid fold, and arranged in a manner that generates two hydrophobic cores that are capped at the end of the chain by a salt bridge. The outer surface of the monomers presents hydrophilic side chains to the solvent. The interface between the monomers of the dimer shows clear contacts between M35 of one molecule and L17 and Q15 of the second. Intermolecular ¹³C–¹⁵N constraints demonstrate that the amyloid fibrils are parallel in register. The RMSD of the backbone structure (Q15–A42) is 0.71 ± 0.12 Å and of all heavy atoms is 1.07 ± 0.08 Å. The structure provides a point of departure for the design of drugs that bind to the fibril surface and therefore interfere with secondary nucleation and for other therapeutic approaches to mitigate Aβ₄₂ aggregation

Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR

Dynamic nuclear polarization (DNP) enhances the signal in solid-state NMR of proteins by transferring polarization from electronic spins to the nuclear spins of interest. Typically, both the protein and an exogenous source of electronic spins, such as a biradical, are either codissolved or suspended and then frozen in a glycerol/water glassy matrix to achieve a homogeneous distribution. While the use of such a matrix protects the protein upon freezing, it also reduces the available sample volume (by ca. a factor of 4 in our experiments) and causes proportional NMR signal loss. Here we demonstrate an alternative approach that does not rely on dispersing the DNP agent in a glassy matrix. We synthesize a new biradical, ToSMTSL, which is based on the known DNP agent TOTAPOL, but also contains a thiol-specific methanethiosulfonate group to allow for incorporating this biradical into a protein in a site-directed manner. ToSMTSL was characterized by EPR and tested for DNP of a heptahelical transmembrane protein, <i>Anabaena</i> sensory rhodopsin (ASR), by covalent modification of solvent-exposed cysteine residues in two ¹⁵N-labeled ASR mutants. DNP enhancements were measured at 400 MHz/263 GHz NMR/EPR frequencies for a series of samples prepared in deuterated and protonated buffers and with varied biradical/protein ratios. While the maximum DNP enhancement of 15 obtained in these samples is comparable to that observed for an ASR sample cosuspended with ∼17 mM TOTAPOL in a glycerol-<i>d</i>₈/D₂O/H₂O matrix, the achievable sensitivity would be 4-fold greater due to the gain in the filling factor. We anticipate that the DNP enhancements could be further improved by optimizing the biradical structure. The use of covalently attached biradicals would broaden the applicability of DNP NMR to structural studies of proteins

Higher Order Amyloid Fibril Structure by MAS NMR and DNP Spectroscopy

Protein magic angle spinning (MAS) NMR spectroscopy has generated structural models of several amyloid fibril systems, thus providing valuable information regarding the forces and interactions that confer the extraordinary stability of the amyloid architecture. Despite these advances, however, obtaining atomic resolution information describing the higher levels of structural organization within the fibrils remains a significant challenge. Here, we detail MAS NMR experiments and sample labeling schemes designed specifically to probe such higher order amyloid structure, and we have applied them to the fibrils formed by an eleven-residue segment of the amyloidogenic protein transthyretin (TTR(105–115)). These experiments have allowed us to define unambiguously not only the arrangement of the peptide β-strands into β-sheets but also the β-sheet interfaces within each protofilament, and in addition to identify the nature of the protofilament-to-protofilament contacts that lead to the formation of the complete fibril. Our efforts have resulted in 111 quantitative distance and torsion angle restraints (10 per residue) that describe the various levels of structure organization. The experiments benefited extensively from the use of dynamic nuclear polarization (DNP), which in some cases allowed us to shorten the data acquisition time from days to hours and to improve significantly the signal-to-noise ratios of the spectra. The β-sheet interface and protofilament interactions identified here revealed local variations in the structure that result in multiple peaks for the exposed N- and C-termini of the peptide and in inhomogeneous line-broadening for the residues buried within the interior of the fibrils