High-frequency high-power DNP/EPR spectrometer operating at 7 T magnetic field.

One of the most essential prerequisites for the development of pulse Dynamic Nuclear Polarization (DNP) is the ability to generate high-power coherent mm-wave pulses at the electron precession frequencies corresponding to the magnetic fields of modern high-resolution NMR spectrometers. As a major step towards achieving this goal, an Extended Interaction Klystron (EIK) pulse amplifier custom-built by the Communications and Power Industries, Inc. and producing up to 140 W at 197.8 GHz, was integrated with in-house built NMR/DNP/EPR spectrometer operating at 7 T magnetic field. The spectrometer employs a Thomas Keating, Ltd. quasioptical bridge to direct mm-waves into a homebuilt DNP probe incorporating photonic bandgap (PBG) resonators to further boost electronic B1e fields. Three-pulse electron spin echo nutation experiments were employed to characterize the B1e fields at the sample by operating the homodyne 198 GHz bridge in an induction mode. Room-temperature experiments with a single-crystal high-pressure, high-temperature (HPHT) diamond and a polystyrene film doped with BDPA radical yielded < 9 ns π/2 pulses at ca. 50 W specified EIK output at the corresponding resonance frequencies and the PBG resonator quality factor of Q≈300. DNP experiments carried out in a "gated" mode by supplying 20 µs mm-wave pulses every 1 ms yielded 13C solid-effect DNP with gains up to 20 for the polystyrene-BDPA sample at natural 13C abundance. For a single-crystal HPHT diamond, the gated DNP mode yielded almost the same 13C enhancement as a low-power continuous wave (CW) mode at 0.4 W, whereas no DNP effect was observed for the BDPA/polystyrene sample in the latter case. To illustrate the versatility of our upgraded DNP spectrometer, room-temperature Overhauser DNP enhancements of 7-14 for 31P NMR signal were demonstrated using a liquid droplet of 1 M tri-phenyl phosphine co-dissolved with 100 mM of BDPA in toluene­d8.

Characterization of photonic band resonators for DNP NMR of thin film samples at 7 T magnetic field.

Polarization of nuclear spins via Dynamic Nuclear Polarization (DNP) relies on generating sufficiently high mm-wave B1e fields over the sample, which could be achieved by developing suitable resonance structures. Recently, we have introduced one-dimensional photonic band gap (1D PBG) resonators for DNP and reported on prototype devices operating at ca. 200 GHz electron resonance frequency. Here we systematically compare the performance of five (5) PBG resonators constructed from various alternating dielectric layers by monitoring the DNP effect on natural-abundance 13C spins in synthetic diamond microparticles embedded into a commercial polyester-based lapping film of just 3 mil (76 µm) thickness. An odd-numbered configuration of dielectric layers for 1D PBG resonator was introduced to achieve further B1e enhancements. Among the PBG configurations tested, combinations of high-ε perovskite LiTaO3 together with AlN as well as AlN with optical quartz wafers have resulted in ca. 40 to over 50- fold gains in the average mm-wave power over the sample vs. the mirror-only configuration. The results are rationalized in terms of the electromagnetic energy distribution inside the resonators obtained analytically and from COMSOL simulations. It was found that average of B1e2 over the sample strongly depends on the arrangement of the dielectric layers that are the closest to the sample, which favors odd-numbered PBG resonator configurations for their use in DNP.

A biradical-tagged phospholipid as a polarizing agent for solid-state MAS Dynamic Nuclear Polarization NMR of membrane proteins.

A novel Dynamic Nuclear Polarization (DNP) NMR polarizing agent ToSMTSL-PTE representing a phospholipid with a biradical TOTAPOL tethered to the polar head group has been synthesized, characterized, and employed to enhance solid-state Nuclear Magnetic Resonance (SSNMR) signal of a lipid-reconstituted integral membrane protein proteorhodopsin (PR). A matrix-free PR formulation for DNP improved the absolute sensitivity of NMR signal by a factor of ca. 4 compared to a conventional preparation with TOTAPOL dispersed in a glassy glycerol/water matrix. DNP enhancements measured at 400 MHz/263 GHz and 600 MHz/395 GHz showed a strong field dependence but remained moderate at both fields, and comparable to those obtained for PR covalently modified with ToSMTSL. Additional continuous wave (CW) X-band electron paramagnetic resonance (EPR) experiments with ToSMTSL-PTE in solutions and in lipid bilayers revealed that an unfavorable conformational change of the linker connecting mononitroxides could be one of the reasons for moderate DNP enhancements. Further, differential scanning calorimetry (DSC) and CW EPR experiments indicated an inhomogeneous distribution and/or a possibility of a partial aggregation of ToSMTSL-PTE in DMPC:DMPA bilayers when the concentration of the polarizing agent was increased to 20 mol% to maximize the DNP enhancement. Thus, conformational changes and an inhomogeneous distribution of the lipid-based biradicals in lipid bilayers emerged as important factors to consider for further development of this matrix-free approach for DNP of membrane proteins.

Enhancing sensitivity of Double Electron-Electron Resonance (DEER) by using Relaxation-Optimized Acquisition Length Distribution (RELOAD) scheme.

Over the past decades pulsed electron-electron double resonance (PELDOR), often called double electron-electron resonance (DEER), became one of the major spectroscopic tools for measurements of nanometer-scale distances and distance distributions in non-crystalline biological and chemical systems. The method is based on detecting the amplitude of the primary (3-pulse DEER) or refocused (4-pulse DEER) spin echo for the so-called "observer" spins when the other spins coupled to the former by a dipolar interaction are flipped by a "pump" pulse at another EPR frequency. While the timing of the pump pulse is varied in steps, the positions of the observer pulses are typically fixed. For such a detection scheme the total length of the observer pulse train and the electron spin memory time determine the amplitude of the detected echo signal. Usually, the distance range considerations in DEER experiments dictate the total length of the observer pulse train to exceed the phase memory time by a factor of few and this leads to a dramatic loss of the signal-to-noise ratio (SNR). While the acquisition of the DEER signal seems to be irrational under such conditions, it is currently the preferred way to conduct DEER because of an effective filtering out of all other unwanted interactions. Here we propose a novel albeit simple approach to improve DEER sensitivity and decrease data acquisition time by introducing the signal acquisition scheme based on RELaxation Optimized Acquisition (Length) Distribution (DEER-RELOAD). In DEER-RELOAD the dipolar phase evolution signal is acquired in multiple segments in which the observer pulses are fixed at the positions to optimize SNR just for that specific segment. The length of the segment is chosen to maximize the signal acquisition efficiency according the phase relaxation properties of the spin system. The total DEER trace is then obtained by "stitching" the multiple segments into a one continuous trace. The utility of the DEER-RELOAD acquisition scheme has been demonstrated on an example of the standard 4-pulse DEER sequence applied to two membrane protein complexes labeled with nitroxides. While theoretical gains from the DEER-RELOAD scheme increase with the number of stitched segments, in practice, even dividing the acquisition of the DEER trace into two segments may improve SNR by a factor of >3, as it has been demonstrated for one of these two membrane proteins.

Multi-resonant photonic band-gap/saddle coil DNP probehead for static solid state NMR of microliter volume samples.

The most critical condition for performing Dynamic Nuclear Polarization (DNP) NMR experiments is achieving sufficiently high electronic B1e fields over the sample at the matched EPR frequencies, which for modern high-resolution NMR instruments fall into the millimeter wave (mmW) range. Typically, mmWs are generated by powerful gyrotrons and/or extended interaction klystrons (EIKs) sources and then focused onto the sample by dielectric lenses. However, further development of DNP methods including new DNP pulse sequences may require B1e fields higher than one could achieve with the current mmW technology. In order to address the challenge of significantly enhancing the mmW field at the sample, we have constructed and tested one-dimensional photonic band-gap (PBG) mmW resonator that was incorporated inside a double-tuned radiofrequency (rf) NMR saddle coil. The photonic crystal is formed by stacking ceramic discs with alternating high and low dielectric constants and thicknesses of λ/4 or 3λ/4, where λ is the wavelength of the incident mmW field in the corresponding dielectric material. When the mmW frequency is within the band gap of the photonic crystal, a defect created in the middle of the crystal confines the mmW energy, thus forming a resonant structure. An aluminum mirror in the middle of the defect has been used to substitute one-half of the structure with its mirror image in order to reduce the resonator size and simplify its tuning. The latter is achieved by adjusting the width of the defect by moving the aluminum mirror with respect to the dielectric stack using a gear mechanism. The 1D PBG resonator was the key element for constructing a multi-resonant integrated DNP/NMR probehead operating at 190-199 GHz EPR/300 MHz 1H/75.5 MHz 13C NMR frequencies. Initial tests of the multi-resonant DNP/NMR probehead were carried out using a quasioptical mmW  bridge and a Bruker Biospin Avance II spectrometer equipped with a standard Bruker 7 T wide-bore 89 mm magnet parked at 300.13 MHz 1H NMR frequency. The mmW bridge built with all solid-state active components allows for the frequency tuning between ca. 190 and ca. 199 GHz with the output power up to 27 dBm (0.5 W) at 192 GHz and up to 23 dBm (0.2 W) at 197.5 GHz. Room temperature DNP experiments with a synthetic single crystal high-pressure high-temperature (HPHT) diamond (0.3 × 0.3 × 3.0 mm3) demonstrated dramatic 1500-fold enhancement of 13C natural abundance NMR signal at full incident mmW power. Significant 13C DNP enhancement (of about 90) have been obtained at incident mmW powers of as low as <100 µW. Further tests of the resonator performance have been carried out with a thin (ca. 100 µm thickness) composite polystyrene-microdiamond film by controlling the average mmW power at the optimal DNP conditions via a gated mode of operation. From these experiments, the PBG resonator with loaded Q ≃ 250 and finesse F≈75 provides up to 12-fold or 11 db gain in the average mmW power vs. the non-resonant probehead configuration employing only a reflective mirror.

A comparative study of the nanoscale and macroscale tribological attributes of alumina and stainless steel surfaces immersed in aqueous suspensions of positively or negatively charged nanodiamonds.

This article reports a comparative study of the nanoscale and macroscale tribological attributes of alumina and stainless steel surfaces immersed in aqueous suspensions of positively (hydroxylated) or negatively (carboxylated) charged nanodiamonds (ND). Immersion in -ND suspensions resulted in a decrease in the macroscopic friction coefficients to values in the range 0.05-0.1 for both stainless steel and alumina, while +ND suspensions yielded an increase in friction for stainless steel contacts but little to no increase for alumina contacts. Quartz crystal microbalance (QCM), atomic force microscopy (AFM) and scanning electron microscopy (SEM) measurements were employed to assess nanoparticle uptake, surface polishing, and resistance to solid-liquid interfacial shear motion. The QCM studies revealed abrupt changes to the surfaces of both alumina and stainless steel upon injection of -ND into the surrounding water environment that are consistent with strong attachment of NDs and/or chemical changes to the surfaces. AFM images of the surfaces indicated slight increases in the surface roughness upon an exposure to both +ND and -ND suspensions. A suggested mechanism for these observations is that carboxylated -NDs from aqueous suspensions are forming robust lubricious deposits on stainless and alumina surfaces that enable gliding of the surfaces through the -ND suspensions with relatively low resistance to shear. In contrast, +ND suspensions are failing to improve tribological performance for either of the surfaces and may have abraded existing protective boundary layers in the case of stainless steel contacts. This study therefore reveals atomic scale details associated with systems that exhibit starkly different macroscale tribological properties, enabling future efforts to predict and design complex lubricant interfaces.

Spin Probe Multi-Frequency EPR Study of Unprocessed Cotton Fibers.

Known since the ancient times, cotton continues to be one of the essential materials for the human civilization. Cotton fibers are almost pure cellulose and contain both crystalline and amorphous nanodomains with different physicochemical properties. While understanding of interactions between the individual cellulose chains within the crystalline phase is important from a perspective of mechanical properties, studies of the amorphous phase lead to characterization of the essential transport parameters, such as solvent diffusion, dyeing, drug release, and toxin absorption, as well as more complex processes of enzymatic degradation. Here, we describe the use of spin probe electron paramagnetic resonance methods to study local polarity and heterogeneous viscosity of two types of unprocessed cotton fibers, G. hirsutum and G. barbadense, harvested in the State of North Carolina, USA. These fibers were loaded with two small molecule nitroxide probes that differ in polarity-Tempo and its more hydrophilic derivative Tempol-using a series of polar and non-polar solvents. The electron paramagnetic resonance spectra of the nitroxide-loaded cotton fibers were analyzed both semi-empirically and by least-squares simulations using a rigorous stochastic theory of electron paramagnetic resonance spectra developed by Freed and coworkers. A software package and least-squares fitting protocols were developed to carry out automatic simulations of multi-component electron paramagnetic resonance spectra in both first-derivative and the absorption forms at multiple resonance frequencies such as X-band (9.5 GHz) and W-band (94.3 GHz). The results are compared with the preceding electron paramagnetic resonance spin probe studies of a commercial bleached cotton sheeting carried out by Batchelor and coworkers. One of the results of this study is a demonstration of a co-existence of cellulose nanodomains with different physicochemical properties such as polarity and microviscosity that are affected by solvents and temperature. Spin labeling studies also revealed a macroscopic heterogeneity in the domain distribution along the cotton fibers and a critical role the cuticular layer is playing as a barrier for spin probe penetration. Finally but not lastly, the simultaneous multi-component least-squares simulation method of electron paramagnetic resonance spectra acquired at different resonant frequencies and the display forms (e.g., absorption and first-derivative displays) and the strategy of spectral parameter sharing could be potentially applicable to other heterogeneous biological systems in addition to the cotton fibers studies here.

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, Anabaena sensory rhodopsin (ASR), by covalent modification of solvent-exposed cysteine residues in two (15)N-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-d8/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.

Nanotube array method for studying lipid-induced conformational changes of a membrane protein by solid-state NMR.

Anodic aluminum oxide substrates with macroscopically aligned homogeneous nanopores of 80 nm in diameter enable two-dimensional, solid-state nuclear magnetic resonance studies of lipid-induced conformational changes of uniformly (15)N-labeled Pf1 coat protein in native-like bilayers. The Pf1 helix tilt angles in bilayers composed of two different lipids are not entirely governed by the membrane thickness but could be rationalized by hydrophobic interactions of lysines at the bilayer interface. The anodic aluminum oxide alignment method is applicable to a broader repertoire of lipids versus bicelle bilayer mimetics currently employed in solid-state nuclear magnetic resonance of oriented samples, thus allowing for elucidation of the role played by lipids in shaping membrane proteins.

EPR assessment of protein sites for incorporation of Gd(III) MRI contrast labels.

We have engineered apolipoprotein A-I (apoA-I), a major protein constituent of high-density lipoprotein (HDL), to contain DOTA-chelated Gd(III) as an MRI contrast agent for the purpose of imaging reconstituted HDL (rHDL) biodistribution, metabolism and regulation in vivo. This protein contrast agent was obtained by attaching the thiol-reactive Gd[MTS-ADO3A] label at Cys residues replaced at four distinct positions (52, 55, 76 and 80) in apoA-I. MRI of infused mice previously showed that the Gd-labeled apoA-I migrates to both the liver and the kidney, the organs responsible for HDL catabolism; however, the contrast properties of apoA-I are superior when the ADO3A moiety is located at position 55, compared with the protein labeled at positions 52, 76 or 80. It is shown here that continuous wave X-band (9 GHz) electron paramagnetic resonance (EPR) spectroscopy is capable of detecting differences in the Gd(III) signal when comparing the labeled protein in the lipid-free with the rHDL state. Furthermore, the values of NMR relaxivity obtained for labeled variants in both the lipid-free and rHDL states correlate to the product of the X-band Gd(III) spectral width and the collision frequency between a nitroxide spin label and a polar relaxation agent. Consistent with its superior relaxivity measured by NMR, the rHDL-associated apoA-I containing the Gd[MTS-ADO3A] probe attached to position 55 displays favorable dynamic and water accessibility properties as determined by X-band EPR. While room temperature EPR requires >1 m m Gd(III)-labeled and only >10 µ m nitroxide-labeled protein to resolve the spectrum, the volume requirement is exceptionally low (~5 µl). Thus, X-band EPR provides a practical assessment for the suitability of imaging candidates containing the site-directed ADO3A contrast probe.

Structural changes of UHMWPE after e-beam irradiation and thermal treatment.

Ultra-high molecular weight polyethylene (UHMWPE) was irradiated with accelerated electrons (1 MeV in air) using high dose rates (> 25 kGy/min) and thin specimens (thickness 1 mm). Parts of the specimens were remelted (200 degrees C for 10 min; 150 degrees C for 0, 2, 10, 30, 60 min). All specimens were stored in nitrogen in the dark at 5 degrees C. Supermolecular structure, extent of crosslinking, oxidative degradation, and macroradical content were studied by a number of methods (SAXS, WAXS, SEM, DSC, FTIR, ESR, TGA, solubility experiments, image analysis). The results obtained with irradiated samples were compared with those obtained with irradiated and remelted samples. It was confirmed that crosslinking predominates over chain scission at very high dose rates, even if the irradiation is performed in air. Discrepancies concerning supermolecular structure changes in UHMWPE after irradiation and thermal treatment, found in various studies in the literature, are discussed. A simple model, which describes and explains all supermolecular structure changes, is introduced. An effective way of eliminating residual macroradicals in UHMWPE is proposed.