Search

The sensitivity enhancements available from dynamic nuclear polarization (DNP) are rapidly reshaping the research landscape and expanding the field of nuclear magnetic resonance (NMR) spectroscopy as a tool for solving complex chemical and structural problems. The past decade has seen considerable advances in this burgeoning method, while efforts to further improve its capabilities continue along many avenues. In this report, we examine the influence of static magnetic field strength and temperature on the reported (1)H DNP enhancements from three conventional organic biradicals: TOTAPOL, AMUPol, and SPIROPOL. In contrast to the conventional wisdom, our findings show that at liquid nitrogen temperatures and 700 MHz/460.5 GHz, these three bisnitroxides all provide similar (1)H DNP enhancements, ε ≈ 60. Furthermore, we investigate the influence of temperature, microwave power, magnetic field strength, and protein sample deuteration on the NMR experimental results.

This paper presents a study of coherent dynamic nuclear polarization (DNP) using frequency swept pulses at 94 GHz which optimize the polarization transfer efficiency. Accordingly, an enhancement ℇ∼496 was observed using 10mM trityl-OX063 as the polarizing agent in a standard d8-glycerol:D2O:H2O :: 6:3:1 glassing matrix at 70K. At present this is the largest DNP enhancement reported at this microwave frequency and temperature. Furthermore, the frequency swept pulses enhance the nuclear magnetic resonance (NMR) signal and reduce the recycle delay, accelerating the NMR signal acquisition.

Sensitive high-resolution multidimensional solid-state NMR method is reported for facile identification of hydrogen-bonded 15N–17O pairs in peptides.

Oxygen is an integral component of proteins but remains sparsely studied because its only NMR active isotope, (17)O, has low sensitivity, low resolution, and large quadrupolar couplings. These issues are addressed here with efficient isotopic labeling, high magnetic fields, fast sample spinning, and (1)H detection in conjunction with multidimensional experiments to observe oxygen sites specific to each amino acid residue. Notably, cross-polarization at high sample spinning frequencies provides efficient (13)C ↔ (17)O polarization transfer. The use of (17)O for initial polarization is found to provide better sensitivity per unit time compared to (1)H. Sharp isotropic (17)O peaks are obtained by using a low-power multiple-quantum sequence, which in turn allows extraction of quadrupolar parameters for each oxygen site. Finally, the potential to determine sequential assignments and long-range distance restraints is demonstrated by using 3D (1)H/(13)C/(17)O experiments, suggesting that such methods can become an essential tool for biomolecular structure determination.

We recently used selective (2)H labeling of BDPA to investigate the Overhauser Effect (OE) dynamic nuclear polarization (DNP) mechanism in insulating solids doped with 1,3-bis(diphenylene)-2-phenylallyl (BDPA), and established that the α and γ (1)H spins on the fluorene rings are responsible for generating a zero quantum (ZQ) mediated positive bulk polarization. Here, we establish that the phenyl (1)H spins relax via double-quantum (DQ) processes and therefore contribute negative enhancements which attenuate the OE-DNP. With measurements at different magnetic field strengths, we show that phenyl-d(5)-BDPA offers >50% improvement in OE-DNP enhancement compared to h(21)-BDPA attaining a maximum of ~ 90 at 14.1 T and 5 kHz MAS, the highest observed OE-DNP enhancement to date under these conditions. The approach may be utilized to optimize other polarizing agents exhibiting an OE, an important DNP mechanism with a favorable field and spinning frequency dependence.

Recent structural investigation of amyloid filaments extracted from human patients demonstrated that the ex vivo filaments associated with different disease phenotypes adopt diverse molecular conformations, which are different from those of in vitro amyloid filaments. A very recent cryo-EM structural study also revealed that ex vivo α-synuclein filaments extracted from multiple system atrophy patients adopt distinct molecular structures from those of in vitro α-synuclein filaments, suggesting the presence of co-factors for α-synuclein aggregation in vivo. Here, we report structural characterizations of α-synuclein filaments formed in the presence of a potential co-factor, tau, using cryo-EM and solid-state NMR. Our cryo-EM structure of the tau-promoted α-synuclein filaments reveals some similarities to one of the previously reported polymorphs of in vitro α-synuclein filaments in the core region, while illustrating distinct conformations in the N- and C-terminal regions. The structural study highlights the conformational plasticity of α-synuclein filaments and the importance of the co-factors, requiring additional structural investigation of not only more ex vivo α-synuclein filaments, but also in vitro α-synuclein filaments formed in the presence of diverse co-factors. The comparative structural analyses will help better understand molecular basis of diverse structures of α-synuclein filaments and possible relevance of each structure to the disease phenotype.

Recent advancements in quantum sensing have sparked transformative detection technologies with high sensitivity, precision, and spatial resolution. Owing to their atomic-level tunability, molecular qubits and ensembles thereof are promising candidates for sensing chemical analytes. Here, we show quantum sensing of lithium ions in solution at room temperature with an ensemble of organic radicals integrated in a microporous metal‒organic framework (MOF). The organic radicals exhibit electron spin coherence and microwave addressability at room temperature, thus behaving as qubits. The high surface area of the MOF promotes accessibility of the guest analytes to the organic qubits, enabling unambiguous identification of lithium ions and quantitative measurement of their concentration through relaxometric and hyperfine spectroscopic methods based on electron paramagnetic resonance (EPR) spectroscopy. The sensing principle presented in this work is applicable to other metal ions with nonzero nuclear spin.

Oxygen is an integral component of proteins and nucleic acids, but remains sparsely studied in such samples because its only NMR active isotope, 17O, is a low sensitivity and resolution species. These properties are a consequence of its low natural abundance (0.039%) and the fact that 17O is a S = 5/2 nuclide with large quadrupolar couplings (6-11 MHz). In this work, we address these issues with efficient isotopic labeling, high magnetic fields, fast magic-angle spinning and indirect 1H detection. This combination of refinements, in conjunction with multidimensional heteronuclear correlation experiments, improves sensitivity and permits observation of oxygen sites specific to each amino acid residue in a model dipeptide sample in a manner consistent with the goal of high resolution. In particular, double-quantum cross-polarization at high sample spinning frequencies is found to provide efficient polarization transfer between 13C and 17O nuclei. Notably, the use of 17O as the initial source of polarization for experiments, as opposed to 1H, is found to be advantageous in terms of sensitivity per unit time due to the short 17O T1 relaxation. Additionally, the second-order quadrupolar broadening in the 17O dimension is averaged by incorporation of a low-power multiple-quantum sequence to yield sharp isotropic peaks. Comparison of isotropic and anisotropic 17O spectra allows extraction of quadrupolar parameters for each oxygen site. Finally, the high 17O resolution obtained is used in 3D experiments in combination with 13C polarization transfers and subsequent 1H detection to demonstrate the potential to determine sequential assignments and long range distance restraints. Collectively, these results suggest that 17O correlation spectroscopy can become an essential tool in the repertoire of techniques for biomolecular structure determination with the backbone 17O that has not yet been fully utilized.

This paper presents a theory describing the dynamic nuclear polarization (DNP) process associated with an arbitrary frequency swept microwave pulse. The theory is utilized to explain the integrated solid effect (ISE) as well as the newly discovered stretched solid effect (SSE) and adiabatic solid effect (ASE). It is verified with experiments performed at 9.4 GHz (0.34 T) on single crystals of naphthalene doped with pentacene-d(14). It is shown that the SSE and ASE can be more efficient than the ISE. Furthermore, the theory predicts that the efficiency of the SSE improves at high magnetic fields, where the EPR line width is small compared to the nuclear Larmor frequency. In addition, we show that the ISE, SSE, and ASE are based on similar physical principles and we suggest definitions to distinguish among them.

The solid effect (SE) is a two spin dynamic nuclear polarization (DNP) mechanism that enhances the sensitivity in NMR experiments by irradiation of the electron–nuclear spin transitions with continuous wave (CW) microwaves at ω(0S) ± ω(0I), where ω(0S) and ω(0I) are electron and nuclear Larmor frequencies, respectively. Using trityl (OX063), dispersed in a 60/40 glycerol/water mixture at 80 K, as a polarizing agent, we show here that application of a chirped microwave pulse, with a bandwidth comparable to the EPR line width applied at the SE matching condition, improves the enhancement by a factor of 2.4 over the CW method. Furthermore, the chirped pulse yields an enhancement that is ~20% larger than obtained with the ramped-amplitude NOVEL (RA-NOVEL), which to date has achieved the largest enhancements in time domain DNP experiments. Numerical simulations suggest that the spins follow an adiabatic trajectory during the polarization transfer; hence, we denote this sequence as an adiabatic solid effect (ASE). We foresee that ASE will be a practical pulsed DNP experiment to be implemented at higher static magnetic fields due to the moderate power requirement. In particular, the ASE uses only 13% of the maximum microwave power required for RA-NOVEL.

Dynamic nuclear polarization (DNP) is an NMR sensitivity enhancement technique that mediates polarization transfer from unpaired electrons to NMR-active nuclei. Despite its success in elucidating important structural information on biological and inorganic materials, the detailed polarization-transfer pathway from the electrons to the nearby and then the bulk solvent nuclei, and finally to the molecules of interest-remains unclear. In particular, the nuclei in the paramagnetic polarizing agent play significant roles in relaying the enhanced NMR polarizations to more remote nuclei. Despite their importance, the direct NMR observation of these nuclei is challenging because of poor sensitivity. Here, we show that a combined DNP and electron decoupling approach can facilitate direct NMR detection of these nuclei. We achieved an ∼80 % improvement in NMR intensity via electron decoupling at 0.35 T and 80 K on trityl radicals. Moreover, we recorded a DNP enhancement factor of urn:x-wiley:09476539:media:chem202202556:chem202202556-math-0001 ∼90 and ∼11 % higher NMR intensity using electron decoupling on paramagnetic metal-organic framework, magnesium hexaoxytriphenylene (MgHOTP MOF).

3D printing has evolved into an invaluable tool for rapid and cost-effective production of intricate parts. In this paper we describe 3D printing and other rapid prototyping methods to fabricate 3.2 mm stators and drive caps for use in magic angle spinning (MAS) NMR experiments. These components can be fabricated with the assistance of computer-aided design (CAD) software and at a fraction of the cost of commercial parts. Additionally, we show that the performance of these 3D printed stators and drive caps is comparable to commercially available systems and that they have significant advantages over their machined counterparts.

Significance Amyloid-β (Aβ) is the subject of intense scrutiny because of its close association with Alzheimer’s disease (AD), which currently afflicts about 50 million people worldwide. The results reported in this manuscript focus on the new possibilities provided by ultrafast magic-angle spinning (MAS) 1H detection and fast-MAS dynamic nuclear polarization (DNP), which have ushered in a new era for NMR-based structural biology, but whose potential has not yet been fully exploited for the structural investigation of complex amyloid assemblies. This work demonstrates the expeditious structural analysis of amyloid fibrils, without requiring preparation of large sample amounts, and sets the stage for future studies of unlabeled AD peptides derived from tissue samples available in limited quantities., Several publications describing high-resolution structures of amyloid-β (Aβ) and other fibrils have demonstrated that magic-angle spinning (MAS) NMR spectroscopy is an ideal tool for studying amyloids at atomic resolution. Nonetheless, MAS NMR suffers from low sensitivity, requiring relatively large amounts of samples and extensive signal acquisition periods, which in turn limits the questions that can be addressed by atomic-level spectroscopic studies. Here, we show that these drawbacks are removed by utilizing two relatively recent additions to the repertoire of MAS NMR experiments—namely, 1H detection and dynamic nuclear polarization (DNP). We show resolved and sensitive two-dimensional (2D) and three-dimensional (3D) correlations obtained on 13C,15N-enriched, and fully protonated samples of M0Aβ1-42 fibrils by high-field 1H-detected NMR at 23.4 T and 18.8 T, and 13C-detected DNP MAS NMR at 18.8 T. These spectra enable nearly complete resonance assignment of the core of M0Aβ1-42 (K16-A42) using submilligram sample quantities, as well as the detection of numerous unambiguous internuclear proximities defining both the structure of the core and the arrangement of the different monomers. An estimate of the sensitivity of the two approaches indicates that the DNP experiments are currently ∼6.5 times more sensitive than 1H detection. These results suggest that 1H detection and DNP may be the spectroscopic approaches of choice for future studies of Aβ and other amyloid systems.

The Overhauser effect (OE), commonly observed in NMR spectra of liquids and conducting solids, was recently discovered in insulating solids doped with the radical 1,3-bisdiphenylene-2-phenylallyl (BDPA). However, the mechanism of polarization transfer in OE-DNP in insulators is yet to be established, but hyperfine coupling of the radical to protons in BDPA has been proposed. In this paper we present a study that addresses the role of hyperfine couplings via the EPR and DNP measurements on some selectively deuterated BDPA radicals synthesized for this purpose. Newly developed synthetic routes enable selective deuteration at orthogonal positions or perdeuteration of the fluorene moieties with (2)H incorporation of >93%. The fluorene moieties were subsequently used to synthesize two octadeuterated BDPA radicals, 1,3-[α,γ-d(8)]-BDPA and 1,3-[β,δ-d(8)]-BDPA, and a BDPA radical with perdeuterated fluorene moieties, 1,3-[α,β,γ,δ-d(16)]-BDPA. In contrast to the strong positive OE enhancement observed in degassed samples of fully protonated h(21)-BDPA (ε ~ +70), perdeuteration of the fluorenes results in a negative enhancement (ε ~ −13), while selective deuteration of α- and γ-positions (a(iso) ~ 5.4 MHz) in BDPA results in a weak negative OE enhancement (ε ~ −1). Furthermore, deuteration of β- and δ-positions (a(iso) ~ 1.2 MHz) results in a positive OE enhancement (ε ~ +36), albeit with a reduced magnitude relative to that observed in fully protonated BDPA. Our results clearly show the role of the hyperfine coupled α and γ (1)H spins in the BDPA radical in determining the dominance of the zero and double-quantum cross-relaxation pathways and the polarization-transfer mechanism to the bulk matrix.

Human calprotectin (CP, S100A8/S100A9 oligomer, MRP8/MRP14 oligomer) is an abundant innate immune protein that contributes to the host metal-withholding response. Its ability to sequester transition metal nutrients from microbial pathogens depends on a complex interplay of Ca(II) binding and self-association, which converts the αβ heterodimeric apo protein into a Ca(II)-bound (αβ)(2) heterotetramer that displays enhanced transition metal affinities, antimicrobial activity, and protease stability. A paucity of structural data on the αβ heterodimer has hampered molecular understanding of how Ca(II) binding enables CP to exert its metal-sequestering innate immune function. We report solution NMR data that reveal how Ca(II) binding affects the structure and dynamics of the CP αβ heterodimer. These studies provide a structural model in which the apo αβ heterodimer undergoes conformational exchange and switches between two states, a tetramerization-incompetent or “inactive” state and a tetramerization-competent or “active” state. Ca(II) binding to the EF-hands of the αβ heterodimer causes the active state to predominate, resulting in self-association and formation of the (αβ)(2) heterotetramer. Moreover, Ca(II) binding causes local and allosteric ordering of the His(3)Asp and His(6) metal-binding sites, respectively. Ca(II) binding to the non-canonical EF-hand of S100A9 positions (A9)D30 and organizes the His(3)Asp site. Remarkably, Ca(II) binding causes allosteric effects in the C-terminal region of helix α(IV) of S100A9, which stabilizes the α-helicity at positions H91 and H95 and thereby organizes the functionally versatile His(6) site. Collectively, this study illuminates the molecular basis for how CP responds to high extracellular Ca(II) concentrations, which enables its metal-sequestering host-defense function.

[Image: see text]

The second isoform of the human voltage dependent anion channel (VDAC2) is a mitochondrial porin that translocates calcium and other metabolites across the outer mitochondrial membrane. VDAC2 has been implicated in cardioprotection and plays a critical role in a unique apoptotic pathway in tumor cells. Despite its medical importance, there have been few biophysical studies of VDAC2 in large part due to the difficulty of obtaining homogeneous preparations of the protein for spectroscopic characterization. Here we present high resolution magic angle spinning nuclear magnetic resonance (NMR) data obtained from homogeneous preparation of human VDAC2 in 2D crystalline lipid bilayers. The excellent resolution in the spectra permit several sequence-specific assignments of the signals for a large portion of the VDAC2 N-terminus and several other residues in two- and three-dimensional heteronuclear correlation experiments. The first 12 residues appear to be dynamic, are not visible in cross polarization experiments, and they are not sufficiently mobile on very fast timescales to be visible in 13C INEPT experiments. A comparison of the NMR spectra of VDAC2 and VDAC1 obtained from highly similar preparations demonstrates that the spectral quality, line shapes and peak dispersion exhibited by the two proteins are nearly identical. This suggests an overall similar dynamic behavior and conformational homogeneity, which is in contrast to two earlier reports that suggested an inherent conformational heterogeneity of VDAC2 in membranes. The current data suggest that the sample preparation and spectroscopic methods are likely applicable to studying other human membrane porins, including human VDAC3, which has not yet been structurally characterized. ©2019, Springer Nature B.V., NIH Grant (EB001960), NIH Grant (EB002026)

The importance of studying site-specific interactions of structurally similar water molecules in complex systems is well known. We demonstrate the ability to resolve four distinct bound water environments within the crystal structure of lanthanum magnesium nitrate hydrate via (17)O solid state nuclear magnetic resonance (NMR) spectroscopy. The approach utilizes high-resolution multi-dimensional (17)O NMR experiments at high magnetic fields (18.8 – 35.2 T) where each individual water environment was resolved. The quadrupolar coupling constants and asymmetry parameters of the (17)O of each water were determined to be between 6.6 and 7.1 MHz, and 0.83 and 0.90. The resolution of the four unique, yet similar, structural waters within a hydrated crystal via (17)O NMR spectroscopy demonstrates the ability to decipher the unique electronic environment of structural water within a single hydrated crystal structure.

We present the results of an experimental pulsed DNP study at 1.2 T (33.5 GHz/51 MHz electron and 1H Larmor frequencies, respectively). The results include a comparison of constant-amplitude NOVEL (CA-NOVEL), ramped-amplitude NOVEL (RA-NOVEL) and the frequency-swept integrated solid effect (FS-ISE) experiments all of which were performed at the NOVEL matching condition, ω 1 S = ω 0 I , where ω 1 S is the electron Rabi frequency and ω 0 I the proton Larmor frequency. To the best of our knowledge, this is the first pulsed DNP study carried out at field higher than X-band (0.35 T) using the NOVEL condition. A combination of high microwave power (∼150 W) and a microwave cavity with a high Q (∼500) allowed us to satisfy the NOVEL matching condition. We also observed stretched solid effect (S2E) contributions in the Zeeman field profiles when chirped pulses are applied. Furthermore, the high quality factor of the cavity limits the concentration of the radical to ∼5 mM and generates a hysteresis in the FS-ISE experiments. Nevertheless, we observe very high DNP enhancements that are comparable to the results at X-band. These promising outcomes suggest the importance of further studies at even higher fields that delineate the instrumentation and methods required for time domain DNP.

Dynamic Nuclear Polarization Simulation Optimized with a Unified Propagator (DNPSOUP) is an open-source numerical software program that models spin dynamics for dynamic nuclear polarization (DNP). The software package utilizes a direct numerical approach using the inhomogeneous master equation to treat the time evolution of spin density operator under coherent Hamiltonians and stochastic relaxation effects. Here we present the details of the theory behind the software, starting from the master equation, and arriving at characteristic operators for any section of density operator time-evolution. We then provide an overview of the DNPSOUP software architecture. The efficacy of the program is demonstrated by simulating DNP field profiles on small spin systems exploiting both continuous wave and time-domain DNP mechanisms. Examples include Zeeman field profiles for the solid effect, Overhauser effect, and cross effect, and microwave field profiles for NOVEL, off-resonance NOVEL, the integrated solid effect, the stretched solid effect, and TOP-DNP. The software should facilitate a better understanding of the DNP process, aid in the design of optimized DNP polarizing agents, and allow us to examine new pulsed DNP methods at conditions that are not currently experimentally accessible, especially at high magnetic fields with high-power microwave pulses.

Recent structural studies of ex vivo amyloid filaments extracted from human patients demonstrated that the ex vivo filaments associated with different disease phenotypes adopt diverse molecular conformations distinct from those in vitro amyloid filaments. A very recent cryo-EM structural study also revealed that ex vivo α-synuclein filaments extracted from multiple system atrophy (MSA) patients adopt quite distinct molecular structures from those of in vitro α-synuclein filaments, suggesting the presence of co-factors for α-synuclein aggregation in vivo. Here, we report structural characterizations of α-synuclein filaments derived by a potential co-factor, tau, using cryo-EM and solid-state NMR. Our cryo-EM structure of the tau-promoted α-synuclein filament at 4.0 Å resolution is somewhat similar to one of the polymorphs of in vitro α-synuclein filaments. However, the N- and C-terminal regions of the tau-promoted α-synuclein filament have different molecular conformations. Our structural studies highlight the conformational plasticity of α-synuclein filaments, requiring additional structural investigation of not only more ex vivo α-synuclein filaments, but also in vitro α-synuclein filaments formed in the presence of diverse co-factors to better understand molecular basis of diverse molecular conformations of α-synuclein filaments.

Amyloid formation of full-length TTR involves dissociation of the native tetramers into misfolded monomers that self-assemble into amyloid. In addition to the full-length TTR, C-terminal fragments including residues 49-127 were also observed in vivo, implying the presence of additional misfolding pathways. It was previously proposed that a proteolytic cleavage might lead to the formation of the C-terminal fragment TTR amyloid. Here, we report mechanistic studies of misfolding and aggregation of a TTR variant (G53A) in the absence and presence of a serine protease. A proteolytic cleavage of G53A in the CD loop (K48 and T49) with agitation promoted TTR misfolding and aggregation, suggesting that the proteolytic cleavage may lead to the aggregation of the C-terminal fragment (residues 49-127). To gain more detailed insights into TTR misfolding promoted by proteolytic cleavage, we investigated structural changes in G53A TTR in the presence and absence of trypsin. Our combined biophysical analyses revealed that the proteolytic cleavage accelerated the formation of spherical small oligomers, which exhibited cytotoxic activities. However, the truncated TTR appeared to maintain native-like structures, rather than the C-terminal fragment (residues 49-127) being released and unfolded from the native state. In addition, our solid-state nuclear magnetic resonance and Fourier transform infrared structural studies showed that the two aggregates derived from the full-length and cleaved TTR exhibited nearly identical molecular structural features, suggesting that the proteolytic cleavage in the CD loop destabilizes the native tetrameric structure and accelerates oligomer formation through a common TTR misfolding and aggregation mechanism rather than through a distinct molecular mechanism. ©2020 American Chemical Society., National Institutes of Health (Grant NS097490)

© 2020 American Chemical Society. Structural characterization of misfolded protein aggregates is essential to understanding the molecular mechanism of protein aggregation associated with various protein misfolding disorders. Here, we report structural analyses of ex vivo transthyretin aggregates extracted from human cardiac tissue. Comparative structural analyses of in vitro and ex vivo transthyretin aggregates using various biophysical techniques revealed that cardiac transthyretin amyloid has structural features similar to those of in vitro transthyretin amyloid. Our solid-state nuclear magnetic resonance studies showed that in vitro amyloid contains extensive nativelike β-sheet structures, while other loop regions including helical structures are disrupted in the amyloid state. These results suggest that transthyretin undergoes a common misfolding and aggregation transition to nativelike aggregation-prone monomers that self-assemble into amyloid precipitates in vitro and in vivo.

In Alzheimer’s disease (AD) and other tauopathies, the cytosolic protein Tau misfolds and forms intracellular aggregates which accumulate within the brain leading to neurodegeneration. Clinical progression is tightly linked to the progressive spread of Tau pathology throughout the brain, and several lines of evidence suggest that Tau aggregates or “seeds” may propagate pathology by spreading from cell to cell in a “prion like” manner. Accordingly, blocking the spread of extracellular seeds with an antibody could be a viable therapeutic approach. However, as the structure of Tau seeds is unknown, it is only possible to rationally design therapeutic Tau antibodies by making a priori assumptions. To avoid this, we developed a robust and quantitative cell based assay and employed an unbiased screening approach to identify the antibody with the highest activity against human Tau seeds. The selected antibody (D), directed to the mid-region of Tau (amino acids 235–250), potently blocked the seeding of human AD Tau and was also fully efficacious against seeds from progressive supranuclear palsy. When we compared this antibody with previously described reference antibodies, we were surprised to find that none of these antibodies showed comparable efficacy against human pathological seeds. Our data highlight the difficulty of predicting antibody accessible epitopes on pathological Tau seeds and question the potential efficacy of some of the Tau antibodies that are currently in clinical development. Electronic supplementary material The online version of this article (10.1007/s00401-018-1911-2) contains supplementary material, which is available to authorized users.

Significance Conversion of ribonucleotides to the 2′-deoxyribonucleotides required for DNA biosynthesis is catalyzed by ribonucleotide reductases (RNRs) via a free-radical mechanism. Known types of RNRs all depend on redox-active transition metals—manganese, iron, or cobalt—for radical initiation. Pathogenic bacteria are challenged by transition metal sequestration and infliction of oxidative stress by their hosts, and the deployment of multiple RNRs with different metal requirements and radical-initiating oxidants is a known bacterial countermeasure. A class I RNR from two bacterial pathogens completely lacks transition metals in its active state and uses a tyrosine-derived dihydroxyphenylalanine radical as its initiator, embodying a novel tactic to combat transition metal- and oxidant-mediated innate immunity and reinforcing bacterial RNRs as potential antibiotic targets., All cells obtain 2′-deoxyribonucleotides for DNA synthesis through the activity of a ribonucleotide reductase (RNR). The class I RNRs found in humans and pathogenic bacteria differ in (i) use of Fe(II), Mn(II), or both for activation of the dinuclear-metallocofactor subunit, β; (ii) reaction of the reduced dimetal center with dioxygen or superoxide for this activation; (iii) requirement (or lack thereof) for a flavoprotein activase, NrdI, to provide the superoxide from O2; and (iv) use of either a stable tyrosyl radical or a high-valent dimetal cluster to initiate each turnover by oxidizing a cysteine residue in the α subunit to a radical (Cys•). The use of manganese by bacterial class I, subclass b-d RNRs, which contrasts with the exclusive use of iron by the eukaryotic Ia enzymes, appears to be a countermeasure of certain pathogens against iron deprivation imposed by their hosts. Here, we report a metal-free type of class I RNR (subclass e) from two human pathogens. The Cys• in its α subunit is generated by a stable, tyrosine-derived dihydroxyphenylalanine radical (DOPA•) in β. The three-electron oxidation producing DOPA• occurs in Escherichia coli only if the β is coexpressed with the NrdI activase encoded adjacently in the pathogen genome. The independence of this new RNR from transition metals, or the requirement for a single metal ion only transiently for activation, may afford the pathogens an even more potent countermeasure against transition metal-directed innate immunity.

The electronic g factor carries highly useful information about the electronic structure of a paramagnetic species, such as spin-orbit coupling and dia- or paramagnetic (de-)shielding due to local fields of surrounding electron pairs. However, in many cases, a near “spin-only” case is observed, in particular for light elements, necessitating accurate and precise measurement of the g factors. Such measurement is typically impeded by a “chicken and egg situation”: internal or external reference standards are used for relative comparison of electron paramagnetic resonance (EPR) Larmor frequencies. However, the g factor of the standard itself usually is subject to a significant uncertainty which directly limits the precision and/or accuracy of the sought after sample g factor. Here, we apply an EPR reference-free approach for determining the g factor of atomic nitrogen trapped within the endohedral fullerene C60:N@C60 in its polycrystalline state by measuring the 1H NMR resonance frequency of dispersing toluene at room temperature. We found a value of g = 2.00204 ( 4 ) with a finally reached relative precision of ∼20 ppm. This accurate measurement allows us to directly compare the electronic properties of N@C60 to those found in atomic nitrogen in the gas phase or trapped in other solid matrices at liquid helium temperature. We conclude that spin-orbit coupling in N@C60 at room temperature is very similar in magnitude and of same sign as found in other inert solid matrices and that interactions between the quartet spin system and the C60 molecular orbitals are thus negligible.

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 M(o) 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.

The structure of two protected amino acids, FMOC-l-leucine and FMOC-l-valine, and a dipeptide, N-acetyl-l-valyl-l-leucine (N-Ac-VL), were studied via one- and two-dimensional solid-state nuclear magnetic resonance (NMR) spectroscopy. Utilizing (17)O magic-angle spinning (MAS) NMR at multiple magnetic fields (17.6–35.2 T/750–1500 MHz for (1)H) the (17)O quadrupolar and chemical shift parameters were determined for the two oxygen sites of each FMOC-protected amino acids and the three distinct oxygen environments of the dipeptide. The one- and two-dimensional, (17)O, (15)N─(17)O, (13)C─(17)O, and (1)H─(17)O double-resonance correlation experiments performed on the uniformly (13)C,(15)N and 70% (17)O-labeled dipeptide prove the attainability of (17)O as a probe for structure studies of biological systems. (15)N─(17)O and (13)C─(17)O distances were measured via one-dimensional REAPDOR and ZF-TEDOR experimental buildup curves and determined to be within 15% of previously reported distances, thus demonstrating the use of (17)O NMR to quantitate interatomic distances in a fully labeled dipeptide. Through-space hydrogen bonding of N-Ac-VL was investigated by a two-dimensional (1)H-detected (17)O R(3)-R-INEPT experiment, furthering the importance of (17)O for studies of structure in biomolecular solids.

Proton-assisted recoupling (PAR) is examined by exploring optimal experimental conditions and magnetization transfer rates in a variety of biologically relevant nuclear spin-systems, including simple amino acids, model peptides, and two proteins–nanocrystalline protein G (GB1), and importantly amyloid beta 1–42 (M(0)Aβ(1–42)) fibrils. A selective PAR protocol, SUBPAR (setting up better proton assisted recoupling), is described to observe magnetization transfer in one-dimensional spectra, which minimizes experiment time (in comparison to two-dimensional experiments) and thereby enables an efficient assessment of optimal PAR conditions for a desired magnetization transfer. In the case of the peptide spin systems, experimental and simulated PAR data sets are compared on a semiquantitative level, thereby elucidating the interactions influencing PAR magnetization transfer and their manifestations in different spin transfer networks. Using the optimum Rabi frequencies determined by SUBPAR, PAR magnetization transfer trajectories (or buildup curves) were recorded and compared to simulated results for short peptides. PAR buildup curves were also recorded for M(0)Aβ(1–42) and examined conjointly with a recent structural model. The majority of salient cross-peak intensities observed in the M(0)Aβ(1–42) PAR spectra are well-modeled with a simple biexponential equation, although the fitting parameters do not show any strong correlation to internuclear distances. Nevertheless, these parameters provide a wealth of invaluable semiquantitative structural constraints for the M(0)Aβ(1–42). The results presented here offer a complete protocol for recording PAR (13)C–(13)C correlation spectra with high-efficiency and using the resulting information in protein structural studies.

A mechanistic understanding of Aβ aggregation and high-resolution structures of Aβ fibrils and oligomers are vital to elucidating relevant details of neurodegeneration in Alzheimer's disease, which will facilitate the rational design of diagnostic and therapeutic protocols. The most detailed and reproducible insights into structure and kinetics have been achieved using Aβ peptides produced by recombinant expression, which results in an additional methionine at the N-terminus. While the length of the C-terminus is well established to have a profound impact on the peptide's aggregation propensity, structure, and neurotoxicity, the impact of the N-terminal methionine on the aggregation pathways and structure is unclear. For this reason, we have developed a protocol to produce recombinant Aβ1-42, sans the N-terminal methionine, using an N-terminal small ubiquitin-like modifier-Aβ1-42 fusion protein in reasonable yield, with which we compared aggregation kinetics with AβM01-42 containing the additional methionine residue. The data revealed that Aβ1-42 and AβM01-42 aggregate with similar rates and by the same mechanism, in which the generation of new aggregates is dominated by secondary nucleation of monomers on the surface of fibrils. We also recorded magic angle spinning nuclear magnetic resonance spectra that demonstrated that excellent spectral resolution is maintained with both AβM01-42 and Aβ1-42 and that the chemical shifts are virtually identical in dipolar recoupling experiments that provide information about rigid residues. Collectively, these results indicate that the structure of the fibril core is unaffected by N-terminal methionine. This is consistent with the recent structures of AβM01-42 in which M0 is located at the terminus of a disordered 14-amino acid N-terminal tail.

Chloride ions play important roles in many chemical and biological processes. This paper investigates the possibility of localizing 35Cl nuclei using solid-state NMR. It demonstrates that distances shorter than 3.8A, between 13C atoms and 35Cl atoms in 10% uniformly labeled 13C L-tyrosine·HCl and natural abundance Glycine·HCl can be measured using rotational-echo (adiabatic passage) double-resonance (RE(AP)DOR). Furthermore the effect of quadrupolar interaction on the REDOR/REAPDOR experiment is quantified. The dephasing curve is plotted in a three dimensional chart as a function of the dephasing time and of the strength of quadrupolar interaction felt by each orientation. During spinning each orientation feels a quadrupolar interaction that varies in time, and therefore at each moment in time we reorder the crystallite orientations as a function of their contribution to the dephasing curve. In this way the effect of quadrupolar interaction on the dipolar dephasing curve can be fitted with a polynomial function. The numerical investigation performed allows us to generate REDOR/REAPDOR curves which are then used to simulate the experimental data.

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 13C’s and 15N’s. Similarly, the 15N signal from Ala-NH3+ disappears at ~ 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 ~95 K, while all other 13C’s 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.

We demonstrate a novel 3D NNC magic angle spinning (MAS) NMR experiment that generates (15)N-(15)N internuclear contacts in protein systems using an optimized (15)N-(15)N proton assisted recoupling (PAR) mixing period and a (13)C dimension for improved resolution. The optimized PAR condition permits the acquisition of high signal-to-noise 3D data that enables backbone chemical shift assignments using a strategy that is complementary to current schemes. The spectra can also provide distance constraints. The utility of the experiment is demonstrated on an M(0)Ab(1–42) fibril sample that yields high-quality data that is readily assigned and interpreted. The 3D NNC experiment therefore provides a powerful platform for solid-state protein studies and is broadly applicable to a variety of systems and experimental conditions.

We report the design and experimental demonstration of a frequency tunable terahertz gyrotron at 527 GHz built for an 800-MHz dynamic nuclear polarization enhanced nuclear magnetic resonance (DNP-NMR) spectrometer. The gyrotron is designed at the second harmonic ( $\omega = {2}\omega _{c}$ ) of the electron cyclotron frequency. It produces up to 9.3-W continuous microwave (CW) power at 527.2-GHz frequency using a diode type electron gun operating at ${V} = {16.65}$ kV, ${I}_{b} = {110}$ mA in a TE11,2,1 mode, corresponding to an efficiency of ~0.5%. The gyrotron is tunable within ~0.4 GHz by combining voltage and magnetic field tuning. The gyrotron has an internal mode converter that produces a Gaussian-like beam that couples to the HE11 mode of an internal 12-mm i.d. corrugated waveguide periscope assembly leading up to the output window. An external corrugated waveguide transmission line system is built including a corrugated taper from 12- to 16-mm i.d. waveguide followed by 3 m of the 16-mm i.d. waveguide The microwave beam profile is measured using a pyroelectric camera showing ~84% HE11 mode content.

We describe the design and construction of a modular, triple-resonance, fully balanced, DNP-MAS probe based on transmission line technology and its integration into a 500 MHz/330 GHz DNP-NMR spectrometer. A novel quantitative probe design and characterization strategy is developed and employed to achieve optimal sensitivity, RF homogeneity and excellent isolation between channels. The resulting three channel HCN probe has a modular design with each individual, swappable module being equipped with connectorized, transmission line ports. This strategy permits attachment of a mating connector that facilitates accurate impedance measurements at these ports and allows characterization and adjustment (e.g. for balancing or tuning/matching) of each component individually. The RF performance of the probe is excellent; for example, the 13C channel attains a Rabi frequency of 280 kHz for a 3.2 mm rotor. In addition, a frequency tunable 330 GHz gyrotron operating at the second harmonic of the electron cyclotron frequency was developed for DNP applications. Careful alignment of the corrugated waveguide led to minimal loss of the microwave power, and an enhancement factor e = 180 was achieved for U-13C urea in the glassy matrix at 80 K. We demonstrated the operation of the system with acquisition of multidimensional spectra of cross-linked lysozyme crystals which are insoluble in glycerol-water mixtures used for DNP and samples of RNA.

A machine learning approach is presented for analyzing complex two-dimensional hyperfine sublevel correlation electron paramagnetic resonance (HYSCORE EPR) spectra with the proficiency of an expert spectroscopist. The computer vision algorithm requires no training on experimental data; rather, all of the spin physics required to interpret the spectra are learned from simulations alone. This approach is therefore applicable even when insufficient experimental data exist to train the algorithm. The neural network is demonstrated to be capable of utilizing the full information content of two-dimensional (14)N HYSCORE spectra to predict the magnetic coupling parameters and their underlying probability distributions that were previously inaccessible. The predicted hyperfine (a, T) and (14)N quadrupole (K, η) coupling constants deviate from the previous manual analyses of the experimental spectra on average by 0.11 MHz, 0.09 MHz, 0.19 MHz, and 0.09, respectively.

The Lewis acidity of isolated framework metal sites in Beta zeolites was characterized with 15N isotopically labeled pyridine adsorption coupled with magic-angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy. The 15N chemical shift of adsorbed pyridine was found to scale with the acid character of both Lewis (Ti, Hf, Zr, Nb, Ta, and Sn) and Brønsted (B, Ga, and Al) acidic heteroatoms. The 15N chemical shift showed a linear correlation with Mulliken electronegativity of the metal center in the order Ti < Hf < Zr < Nb < Ta < Sn < H+. Theoretical calculations using density functional theory (DFT) showed a strong correlation between experimental 15N chemical shift and the calculated metal–nitrogen bond dissociation energy, and revealed the importance of active site reorganization when determining adsorption strength. The relationships found between 15N pyridine chemical shift and intrinsic chemical descriptors of metal framework sites complement adsorption equilibrium data and provide a robust method to characterize, and ultimately optimize, metal-reactant binding and activation for Lewis acid zeolites. Direct 15N MAS NMR detection protocols applied to the Lewis acid–base adducts allowed the differentiation and quantification of framework metal sites in the presence of extraframework oxides, including highly quadrupolar nuclei that are not amenable for quantification with conventional NMR methods.

Polymer gels are often very soft due to their low branch functionality (f) and the inevitable presence of defects (e.g., primary loops or dangling chains). Polymer metal–organic cage (polyMOC) gels are a relatively new class of supramolecular gels with precisely defined junction structures made possible by subcomponent assembly of nanoscale MOCs connected by polymer chains. Herein, we report that variation of the molecular weight and architecture of the polymer component of polyMOCs provides an entry into gels with ultra-high f. For example, materials with f ∼ 9–12, i.e., ∼ 9–12 polymer chains connect each MOC within the gel network, are realized. As a consequence of their increased f, these gels display exemplary mechanical properties at low concentrations (down to 240 μM) of metal ions and only 5.4–5.9 wt % of polymer. Furthermore, X-ray and neutron scattering pointed to an additional level of structural hierarchy that arises from the assembly of M12L24 MOCs into clusters. The relationships between poly...

The paper describes a family of novel recoupling pulse sequences, called three pulse recoupling. These pulse sequences can be employed for both homonuclear and heteronuclear recoupling experiments and are robust to dispersion in chemical shifts and rf-inhomogeneity. These recoupling pulse sequences can be used in design of two-dimensional solid state NMR experiments that use powdered dephased antiphase coherence (γ preparation) to encode chemical shifts in the indirect dimension. Both components of this chemical shift encoded gamma-prepared states can be refocused into inphase coherence by a recoupling element. This helps to achieve sensitivity enhancement in 2D NMR experiments by quadrature detection.

The chemical structure of polarizing agents critically determines the efficiency of dynamic nuclear polarization (DNP). For cross-effect DNP, biradicals are the polarizing agents of choice and the interaction and relative orientation of the two unpaired electrons should be optimal. Both parameters are affected by the molecular structure of the biradical in the frozen glassy matrix that is typically used for DNP/MAS NMR and likely differs from the structure observed with X-ray crystallography. We have determined the conformations of six bis-nitroxide polarizing agents, including the highly efficient AMUPol, in their DNP matrix with EPR spectroscopy at 9.7 GHz, 140 GHz, and 275 GHz. The multi-frequency approach in combination with an advanced fitting routine allows us to reliably extract the interaction and relative orientation of the nitroxide moieties. We compare the structures of six bis-nitroxides to their DNP performance at 500 MHz/330 GHz., Physical Chemistry Chemical Physics, 20 (39), ISSN:1463-9084, ISSN:1463-9076

We investigate a new time domain approach to dynamic nuclear polarization (DNP), the frequency-swept integrated solid effect (FS-ISE), utilizing a high power, broadband 94 GHz (3.35 T) pulse EPR spectrometer. The bandwidth of the spectrometer enabled measurement of the DNP Zeeman frequency/field profile that revealed two dominant polarization mechanisms, the expected ISE, and a recently observed mechanism, the stretched solid effect (S(2)E). At 94 GHz, despite the limitations in the microwave chirp pulse length (10 μs) and the repetition rate (2 kHz), we obtained signal enhancements up to ~70 for the S(2)E and ~50 for the ISE. The results successfully demonstrate the viability of the FS-ISE and S(2)E DNP at a frequency 10 times higher than previous studies. Our results also suggest that these approaches are candidates for implementation at higher magnetic fields.

Continuous-wave (CW) dynamic nuclear polarization (DNP) is now established as a method of choice to enhance the sensitivity in a variety of NMR experiments. Nevertheless, there remains a need for the development of more efficient methods to transfer polarization from electrons to nuclei. Of particular interest are pulsed DNP methods because they enable a rapid and efficient polarization transfer that, in contrast with CW DNP methods, is not attenuated at high magnetic fields. Here we report nuclear spin orientation via electron spin-locking (NOVEL) experiments using the polarizing agent trityl OX063 in glycerol/water at a temperature of 80 K and a magnetic field of 0.34 T. (1)H NMR signal enhancements up to 430 are observed, and the buildup of the local polarization occurs in a few hundred nanoseconds. Thus, NOVEL can efficiently dynamically polarize (1)H atoms in a system that is of general interest to the solid-state DNP NMR community. This is a first, important step toward the general application of pulsed DNP at higher fields.

Gels formed via metal-ligand coordination typically have very low branch functionality, f, as they consist of ∼2-3 polymer chains linked to single metal ions that serve as junctions. Thus, these materials are very soft and unable to withstand network defects such as dangling ends and loops. We report here a new class of gels assembled from polymeric ligands and metal-organic cages (MOCs) as junctions. The resulting 'polyMOC' gels are precisely tunable and may feature increased branch functionality. We show two examples of such polyMOCs: a gel with a low f based on a M2L4 paddlewheel cluster junction and a compositionally isomeric one of higher f based on a M12L24 cage. The latter features large shear moduli, but also a very large number of elastically inactive loop defects that we subsequently exchanged for functional ligands, with no impact on the gel's shear modulus. Such a ligand substitution is not possible in gels of low f, including the M2L4-based polyMOC.