Facing and Overcoming Sensitivity Challenges in Biomolecular NMR Spectroscopy (original) (raw)
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Journal of Biomolecular NMR, 2020
Signal enhancements of up to two orders of magnitude in protein NMR can be achieved by employing HDO as a vector to introduce hyperpolarization into folded or intrinsically disordered proteins. In this approach, hyperpolarized HDO produced by dissolution-dynamic nuclear polarization (D-DNP) is mixed with a protein solution waiting in a high-field NMR spectrometer, whereupon amide proton exchange and nuclear Overhauser effects (NOE) transfer hyperpolarization to the protein and enable acquisition of a signal-enhanced high-resolution spectrum. To date, the use of this strategy has been limited to 1D and 1 H-15 N 2D correlation experiments. Here we introduce 2D 13 C-detected D-DNP, to reduce exchange-induced broadening and other relaxation penalties that can adversely affect proton-detected D-DNP experiments. We also introduce hyperpolarized 3D spectroscopy, opening the possibility of D-DNP studies of larger proteins and IDPs, where assignment and residue-specific investigation may be impeded by spectral crowding. The signal enhancements obtained depend in particular on the rates of chemical and magnetic exchange of the observed residues, thus resulting in non-uniform 'hyperpolarizationselective' signal enhancements. The resulting spectral sparsity, however, makes it possible to resolve and monitor individual amino acids in IDPs of over 200 residues at acquisition times of just over a minute. We apply the proposed experiments to two model systems: the compactly folded protein ubiquitin, and the intrinsically disordered protein (IDP) osteopontin (OPN).
Journal of Magnetic Resonance, 2009
Intermolecular Multiple-Quantum Coherences (iMQCs) can yield interesting NMR information of high potential usefulness in spectroscopy and imaging -provided their associated sensitivity limitations can be overcome. A recent study demonstrated that ex situ dynamic nuclear polarization (DNP) could assist in overcoming sensitivity problems for iMQC-based experiments on 13 C nuclei. In the present work we show that a similar approach is possible when targeting the protons of a hyperpolarized solvent. It was found that although the DNP procedure enhances single-quantum 1 H signals by about 600, which is significantly less than in optimized low-c liquid-state counterparts, the non-linear dependence of iMQC-derived signals on polarization can yield very large enhancements approaching 10 6 . Cleary no practical amount of data averaging can match this kind of sensitivity gains. The fact that DNP endows iMQC-based 1 H NMR spectra with a sensitivity that amply exceeds that of their thermally polarized single-quantum counterpart, is confirmed in a number of simple single-scan 2D imaging experiments.
Dynamic nuclear polarization for sensitivity enhancement in modern solid-state NMR
Progress in Nuclear Magnetic Resonance Spectroscopy
The field of dynamic nuclear polarization has undergone tremendous developments and diversification since its inception more than 6 decades ago. In this review we provide an in-depth overview of the relevant topics involved in DNP-enhanced MAS NMR spectroscopy. This includes the theoretical description of DNP mechanisms as well as of the polarization transfer pathways that can lead to a uniform or selective spreading of polarization between nuclear spins. Furthermore, we cover historical and state-of-the art aspects of dedicated instrumentation, polarizing agents, and optimization techniques for efficient MAS DNP. Finally, we present an extensive overview on applications in the fields of structural biology and materials science, which underlines that MAS DNP has moved far beyond the proof-of-concept stage and has become an important tool for research in these fields.
Chemical Reviews
Solid-state NMR spectroscopy (ssNMR) with magic-angle spinning (MAS) enables the investigation of biological systems within their native context, such as lipid membranes, viral capsid assemblies, and cells. However, such ambitious investigations often suffer from low sensitivity, due to the presence of significant amounts of other molecular species, which reduces the effective concentration of the biomolecule or interaction of interest. Certain investigations requiring the detection of very low concentration species remain unfeasible even with increasing experimental time for signal averaging. By applying dynamic nuclear polarization (DNP) to overcome the sensitivity challenge, the experimental time required can be reduced by orders of magnitude, broadening the feasible scope of applications for biological solid-state NMR. In this review, we outline strategies commonly adopted for biological applications of DNP, indicate ongoing 1 challenges, and present a comprehensive overview of biological investigations where MAS-DNP has led to unique insights.
1H-Detected 13C Photo-CIDNP as a Sensitivity Enhancement Tool in Solution NMR
Journal of the American Chemical Society, 2011
NMR is a powerful yet intrinsically insensitive technique. The applicability of NMR to chemical and biological systems would be substantially extended by new approaches going beyond current signal-to-noise capabilities. Here, we exploit the large enhancements arising from 13 C photochemically induced dynamic nuclear polarization (13 C photo-CIDNP) in solution to improve biomolecular NMR sensitivity in the context of heteronuclear correlation spectroscopy. The 13 C-PRINT pulse sequence presented here involves an initial 13 C nuclear spin polarization via photo-CIDNP followed by conversion to antiphase coherence and transfer to 1 H for detection. We observe substantial enhancements, up to ≫200-fold, relative to the dark (laser off) experiment. Resonances of both side-chain and backbone CH pairs are enhanced for the three aromatic residues Trp, His and Tyr and the Trp-containing σ 32 peptide. The sensitivity of this experiment, defined as signal-to-noise per unit time (S/N) t , is unprecedented in the NMR polarization enhancement literature dealing with polypeptides in solution. Up to a 16-fold larger (S/N) t than the 1 H-13 C SE-HSQC reference sequence is achieved, for the σ 32 peptide. This gain leads to a reduction in data collection time up to 256-fold, highlighting the advantages of 1 H-detected 13 C photo-CIDNP in solution NMR. NMR is an invaluable spectroscopic tool to probe residue-specific molecular properties such as dynamics, folding and noncovalent interactions. To date, this technique has been largely exploited to study native and nonnative states of biomolecules in solution, including peptides, proteins and nucleic acids. 1 Due to either scarce sample solubility, 2 the need to maintain low concentrations to overcome competing processes (e.g., aggregation), or the intrinsically low abundance of the target species in the relevant physiological environment, 3 liquid-state biomolecular NMR samples are often rather dilute, requiring highly sensitive data collection. In addition, real-time kinetic studies of fast events by NMR impose a need for rapid and efficient data collection even in concentrated samples. 4, 5 NMR's low sensitivity stems from marginal nuclear polarization at thermal equilibrium. Enhanced polarization methods tackle this problem by perturbing the thermal equilibrium upon coupling nuclei to other highly polarized quantum states. 6 For instance, unpaired electron polarization is transferred to nuclei in dynamic nuclear polarization (DNP). 7 Parahydrogen reacts with unsaturated bonds to create 1 H-polarized substrates. 9 Nuclear
Journal of Magnetic Resonance, 2014
Thanks to instrumental and theoretical development, notably the access to high-power and high-frequency microwave sources, high-field dynamic nuclear polarization (DNP) on solid-state NMR currently appears as a promising solution to enhance nuclear magnetization in many different types of systems. In magic-angle-spinning DNP experiments, systems of interest are usually dissolved or suspended in glassforming matrices doped with polarizing agents and measured at low temperature (down to $100 K). In this work, we discuss the influence of sample conditions (radical concentration, sample temperature, etc.) on DNP enhancements and various nuclear relaxation times which affect the absolute sensitivity of DNP spectra, especially in multidimensional experiments. Furthermore, DNP-enhanced solid-state NMR experiments performed at 9.4 T are complemented by high-field CW EPR measurements performed at the same magnetic field. Microwave absorption by the DNP glassy matrix is observed even below the glass transition temperature caused by softening of the glass. Shortening of electron relaxation times due to glass softening and its impact in terms of DNP sensitivity is discussed.
Journal of the American Chemical Society, 2012
The characterization of materials by the inherently insensitive method of NMR spectroscopy plays a vital role in chemistry. Increasingly, hyperpolarization is being used to address the sensitivity limitation. Here, by reference to quinoline, we illustrate that the SABRE hyperpolarization technique, which uses para-hydrogen as the source of polarization, enables the rapid completion of a range of NMR measurements. These include the collection of 13 C, 13 C{ 1 H}, and NOE data in addition to more complex 2D COSY, ultrafast 2D COSY and 2D HMBC spectra. The observations are made possible by the use of a flow probe and external sample preparation cell to re-hyperpolarize the substrate between transients, allowing repeat measurements to be made within seconds. The potential benefit of the combination of SABRE and 2D NMR methods for rapid characterization of low-concentration analytes is therefore established.
High-Field Solid-State NMR with Dynamic Nuclear Polarization
Modern Magnetic Resonance, 2017
Microwave induced dynamic nuclear polarization (DNP) can produce hyperpolarization of nuclear spins, leading to significant signal enhancement in NMR. This chapter discusses the contemporary application of DNP for solid-state NMR spectroscopy at high magnetic fields. The main mechanisms and polarizing agents that enable this hyperpolarization are presented, along with more practical aspects such as the effect of decreasing sample temperature and analysing the absolute sensitivity gain from these experiments. Examples of the exploitation of DNP for studies of biomolecules, biominerals, pharmaceuticals, self-assembled organic nanostructures, and mesoporous materials are given as is an outlook as to the future this powerful technique.
Quantification in Hyperpolarized NMR
Quantitative aspects of hyperpolarized NMR are analyzed in the present work, and it is shown theoretically and experimentally that measured " apparent " signal enhancements could deviate significantly from real enhancements of polarization. Expressions are given as a function of spin count to deduce real enhancements from measured " apparent " enhancements, and vice versa. While the findings are of particular relevance to high-field work employing high-Q probes, and to analytical applications of hyperpolarized NMR whose objective is the measurement of spin count, our experiments demonstrate their significance even for low-and moderate-field work with probes of moderate Q-factor. H yperpolarized NMR is currently witnessing vigorous research activity and renewed popularity because it seeks to address the Achilles heel of NMR, viz., poor sensitivity, without seriously compromising its core strength, viz., high resolution. This opens up a number of chemical applications. Perhaps the earliest polarization enhancement technique in NMR was dynamic nuclear polarization (DNP), 1−3 otherwise known as the Overhauser effect (OE, or ODNP), and related techniques in the solid state, including the solid effect (SE). 4 It was subsequently shown that hyperpolarization could be achieved by suitable techniques that harness the high spin order in parahydrogen, yielding parahydrogen induced polarization (PHIP). 5−10 More recently, PHIP has received a significant boost by the avoidance of a chemical reaction across a multiple bond as originally required, relying instead on a catalyst to bind both parahydrogen and the " substrate " species of interest, accomplishing polarization transfer by spin exchange and NMR signal enhancement in the bulk phase by subsequent chemical exchange; this technique is termed signal amplification by reversible exchange (SABRE). 11−13 While signal enhancement was often the prime motivation in early ODNP studies (which were performed in continuous wave (cw) NMR mode), quantitative measurements for elucidation of the mechanism of the Overhauser effect resulted in the recognition that continuous wave ODNP studies reveal a peculiar effect which results in the deviation of the measured (or " apparent ") enhancement from its real value. 14−16 In this report, we argue that this effect could play a vital role in the time domain measurements typical of modern hyperpolarized NMR, including ODNP, as well as SABRE/PHIP. We give a quantitative relation between the apparent and real enhancement factors, which would aid quantitative analysis based on hyperpolarized NMR spectra. We also demonstrate this effect experimentally in pulsed mode at a moderately low NMR frequency. The basic phenomenon that gives rise to " false " enhancements may be termed the Q-enhancement effect. 14−16 It is to be noted especially that this is a fundamental effect that is unrelated to questions of sample transfer, arising as it does from the fact that the probe circuit quality factor, Q, changes with the sample polarization. In simple terms, the NMR probe which houses the sample under investigation has a quality factor Q that is governed by the coil inductance, L 0 , and effective serial resistance, R 0. Near the resonance condition, this is changed substantially by the NMR characteristics of the sample, viz., the radiofrequency susceptibility, χ, of the spin ensemble, and leads to measurable, even strong, effects under conditions of high spin polarization, especially for high-Q probes and high-frequency measurements. The effect may be modeled in a straightforward manner. The NMR coil impedance, Z, is given in SI units by ω ϕ χ ω ω ϕχ ω χ ω = + + = + ′ − ″ + Z L R L R i [1 ()] i [1 (() i ())] 0 0 0 0 0 0 0 0 0 (1) Here i is the unit imaginary, ϕ the filling factor, and ω 0 the angular frequency at which resonance occurs (i.e., ν 0 Hz). The contribution of the real part of the spin susceptibility, χ′, being zero on resonance, we find for the coil impedance on resonance ω ω ϕχ ω = + ″ + Z L L R i () 0 0 0 0 0 0 (2) For the quality factor of the NMR probe, we thus find that it changes from Q = ω 0 L 0 /R 0 far off resonance to Q′ = ω 0 L 0 / [ω 0 L 0 ϕχ″(ω 0)+R 0 ] on resonance. We may write the measured