Evgeny Markhasin

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.

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.

Efficient, balanced, transmission line RF circuits by back propagation of common impedance nodes.

We present a new, efficient strategy for designing fully balanced transmission line RF circuits for solid state NMR probes based on back propagation of common impedance nodes (BPCIN). In this approach, the impedance node phenomenon is the sole means of achieving mutual RF isolation and balance in all RF channels. BPCIN is illustrated using a custom double resonance 3.2 mm MAS probe operating at 500 MHz ((1)H) and 125 MHz ((13)C). When fully optimized, the probe is capable of producing high homogeneity (810°/90° ratios of 86% and 89% for (1)H and (13)C, respectively) and high efficiency (γB1=100 kHz for (1)H and (13)C at 70 W and 180 W of RF input, respectively; up to 360 kHz for (1)H). The probes performance is illustrated by 2D MAS correlation spectra of microcrystals of the tripeptide N-f-MLF-OH and hydrated amyloid fibrils of the protein PI3-SH3.

Low-Temperature Polymorphic Phase Transition in a Crystalline Tripeptide l-Ala-l-Pro-Gly·H2O Revealed by Adiabatic Calorimetry

We demonstrate application of precise adiabatic vacuum calorimetry to observation of phase transition in the tripeptide L-alanyl-L-prolyl-glycine monohydrate (APG) from 6 to 320 K and report the standard thermodynamic properties of the tripeptide in the entire range. Thus, the heat capacity of APG was measured by adiabatic vacuum calorimetry in the above temperature range. The tripeptide exhibits a reversible first-order solid-to-solid phase transition characterized by strong thermal hysteresis. We report the standard thermodynamic characteristics of this transition and show that differential scanning calorimetry can reliably characterize the observed phase transition with <5 mg of the sample. Additionally, the standard entropy of formation from the elemental substances and the standard entropy of hypothetical reaction of synthesis from the amino acids at 298.15 K were calculated for the studied tripeptide.

Standard Thermodynamic Functions of Tripeptides N‑Formyl‑L‑methionyl‑L‑leucyl‑L‑phenylalaninol and N‑Formyl‑L‑methionyl‑L‑leucyl‑L‑phenylalanine Methyl Ester

The heat capacities of tripeptides N-formyl-l-methionyl-l-leucyl-l-phenylalaninol (N-f-MLF-OH) and N-formyl-l-methionyl-l-leucyl-l-phenylalanine methyl ester (N-f-MLF-OMe) were measured by precision adiabatic vacuum calorimetry over the temperature range from T = (6 to 350) K. The tripeptides were stable over this temperature range, and no phase change, transformation, association, or thermal decomposition was observed. The standard thermodynamic functions: molar heat capacity Cp,m, enthalpy H(T) – H(0), entropy S(T), and Gibbs energy G(T) – H(0) of peptides were calculated over the range from T = (0 to 350) K. The low-temperature (T ≤ 50 K) heat capacities dependencies were analyzed using the Debye’s and the multifractal theories. The standard entropies of formation of peptides at T = 298.15 K were calculated.