@article{nunn_milikisiyants_torelli_monge_delord_shames_meriles_ajoy_smirnov_shenderova_2023, title={Optical and electronic spin properties of fluorescent micro- and nanodiamonds upon prolonged ultrahigh-temperature annealing}, volume={41}, ISSN={["2166-2754"]}, url={https://doi.org/10.1116/6.0002797}, DOI={10.1116/6.0002797}, abstractNote={High-temperature annealing is a promising but still mainly unexplored method for enhancing spin properties of negatively charged nitrogen-vacancy (NV) centers in diamond particles. After high-energy irradiation, the formation of NV centers in diamond particles is typically accomplished via annealing at temperatures in the range of 800–900 °C for 1–2 h to promote vacancy diffusion. Here, we investigate the effects of conventional annealing (900 °C for 2 h) against annealing at a much higher temperature of 1600 °C for the same annealing duration for particles ranging in size from 100 nm to 15 μm using electron paramagnetic resonance and optical characterization. At this high temperature, the vacancy-assisted diffusion of nitrogen can occur. Previously, the annealing of diamond particles at this temperature was performed over short time scales because of concerns of particle graphitization. Our results demonstrate that particles that survive this prolonged 1600 °C annealing show increased NV T1 and T2 electron spin relaxation times in 1 and 15 μm particles, due to the removal of fast relaxing spins. Additionally, this high-temperature annealing also boosts magnetically induced fluorescence contrast of NV centers for particle sizes ranging from 100 nm to 15 μm. At the same time, the content of NV centers is decreased fewfold and reaches a level of <0.5 ppm. The results provide guidance for future studies and the optimization of high-temperature annealing of fluorescent diamond particles for applications relying on the spin properties of NV centers in the host crystals.}, number={4}, journal={JOURNAL OF VACUUM SCIENCE & TECHNOLOGY B}, author={Nunn, Nicholas and Milikisiyants, Sergey and Torelli, Marco D. and Monge, Richard and Delord, Tom and Shames, Alexander I. and Meriles, Carlos A. and Ajoy, Ashok and Smirnov, Alex I. and Shenderova, Olga A.}, year={2023}, month={Jul} }
 @article{nunn_milikisiyants_danilov_torelli_dei cas_zaitsev_shenderova_smirnov_shames_2022, title={Electron irradiation-induced paramagnetic and fluorescent defects in type Ib high pressure-high temperature microcrystalline diamonds and their evolution upon annealing}, volume={132}, ISSN={["1089-7550"]}, url={https://doi.org/10.1063/5.0103313}, DOI={10.1063/5.0103313}, abstractNote={Defects introduced to synthetic type Ib diamond micrometer-size particles by electron-beam irradiation were studied by electron paramagnetic resonance and photoluminescence (PL) spectroscopy as a function of e-beam fluence and post-irradiation thermal annealing. Increasing electron-beam fluence causes a substantial reduction of the substitutional nitrogen (P1) content, accompanied by progressively higher concentrations of paramagnetic negatively charged vacancies (V−) and triplet interstitials (R1/R2). Annealing results in a drastic decrease in the V− and R1/R2 content and an increase in the negatively charged nitrogen-vacancies (NV− or W15). Analysis of PL spectra allows for identification of color centers in the irradiated diamond samples and following their evolution after annealing. These data facilitate understanding of different factors contributing to the formation of color centers in diamond and promote efforts toward controlled engineering of optical centers in fluorescent diamond particles.}, number={7}, journal={JOURNAL OF APPLIED PHYSICS}, author={Nunn, Nicholas and Milikisiyants, Sergey and Danilov, Evgeny O. and Torelli, Marco D. and Dei Cas, Laura and Zaitsev, Alexander and Shenderova, Olga and Smirnov, Alex I. and Shames, Alexander I.}, year={2022}, month={Aug} }
 @article{nevzorov_marek_milikisiyants_smirnov_2021, title={Characterization of photonic band resonators for DNP NMR of thin film samples at 7 T magnetic field}, volume={323}, ISSN={["1096-0856"]}, DOI={10.1016/j.jmr.2020.106893}, abstractNote={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.}, journal={JOURNAL OF MAGNETIC RESONANCE}, author={Nevzorov, Alexander A. and Marek, Antonin and Milikisiyants, Sergey and Smirnov, Alex I.}, year={2021}, month={Feb} }
 @article{chestnut_milikisiyants_chatterjee_kern_smirnov_2021, title={Electronic Structure of the Primary Electron Donor P-700(+center dot) in Photosystem I Studied by Multifrequency HYSCORE Spectroscopy at X- and Q-Band}, volume={125}, ISSN={["1520-5207"]}, url={https://doi.org/10.1021/acs.jpcb.0c09000}, DOI={10.1021/acs.jpcb.0c09000}, abstractNote={The primary electron donor P700 of the photosystem I (PSI) is a heterodimer consisting of two chlorophyll molecules. A series of electron-transfer events immediately following the initial light excitation leads to a stabilization of the positive charge by its cation radical form, P700+•. The electronic structure of P700+• and, in particular, its asymmetry with respect to the two chlorophyll monomers is of fundamental interest and is not fully understood up to this date. Here, we apply multifrequency X- (9 GHz) and Q-band (35 GHz) hyperfine sublevel correlation (HYSCORE) spectroscopy to investigate the electron spin density distribution in the cation radical P700+• of PSI from a thermophilic cyanobacterium Thermosynechococcus elongatus. Six 14N and two 1H distinct nuclei have been resolved in the HYSCORE spectra and parameters of the corresponding nuclear hyperfine and quadrupolar hyperfine interactions were obtained by combining the analysis of HYSCORE spectral features with direct numerical simulations. Based on a close similarity of the nuclear quadrupole tensor parameters, all of the resolved 14N nuclei were assigned to six out of total eight available pyrrole ring nitrogen atoms (i.e., four in each of the chlorophylls), providing direct evidence of spin density delocalization over the both monomers in the heterodimer. Using the obtained experimental values of the 14N electron-nuclear hyperfine interaction parameters, the upper limit of the electron spin density asymmetry parameter is estimated as RA/Bupper = 7.7 ± 0.5, while a tentative assignment of 14N observed in the HYSCORE spectra yields RB/A = 3.1 ± 0.5.}, number={1}, journal={JOURNAL OF PHYSICAL CHEMISTRY B}, publisher={American Chemical Society (ACS)}, author={Chestnut, Melanie M. and Milikisiyants, Sergey and Chatterjee, Ruchira and Kern, Jan and Smirnov, Alex I}, year={2021}, month={Jan}, pages={36–48} }
 @article{saikia_yanez-orozco_qiu_hao_milikisiyants_ou_hamilton_weninger_smirnova_sanabria_et al._2021, title={Integrative structural dynamics probing of the conformational heterogeneity in synaptosomal-associated protein 25}, volume={2}, ISSN={["2666-3864"]}, DOI={10.1016/j.xcrp.2021.100616}, abstractNote={SNAP-25 (synaptosomal-associated protein of 25 kDa) is a prototypical intrinsically disordered protein (IDP) that is unstructured by itself but forms coiled-coil helices in the SNARE complex. With high conformational heterogeneity, detailed structural dynamics of unbound SNAP-25 remain elusive. Here, we report an integrative method to probe the structural dynamics of SNAP-25 by combining replica-exchange discrete molecular dynamics (rxDMD) simulations and label-based experiments at ensemble and single-molecule levels. The rxDMD simulations systematically characterize the coil-to-molten globular transition and reconstruct structural ensemble consistent with prior ensemble experiments. Label-based experiments using Förster resonance energy transfer and double electron-electron resonance further probe the conformational dynamics of SNAP-25. Agreements between simulations and experiments under both ensemble and single-molecule conditions allow us to assign specific helix-coil transitions in SNAP-25 that occur in submillisecond timescales and potentially play a vital role in forming the SNARE complex. We expect that this integrative approach may help further our understanding of IDPs.}, number={11}, journal={CELL REPORTS PHYSICAL SCIENCE}, author={Saikia, Nabanita and Yanez-Orozco, Inna S. and Qiu, Ruoyi and Hao, Pengyu and Milikisiyants, Sergey and Ou, Erkang and Hamilton, George L. and Weninger, Keith R. and Smirnova, Tatyana I and Sanabria, Hugo and et al.}, year={2021}, month={Nov} }
 @article{cha_milikisiyants_davidson_xue_smirnova_smirnov_guo_chang_2020, title={Alternative Reactivity of Leucine 5-Hydroxylase Using an Olefin-Containing Substrate to Construct a Substituted Piperidine Ring}, volume={59}, ISSN={["0006-2960"]}, url={https://doi.org/10.1021/acs.biochem.0c00289}, DOI={10.1021/acs.biochem.0c00289}, abstractNote={Applying enzymatic reactions to produce useful molecules is a central focus of chemical biology. Iron and 2-oxoglutarate (Fe/2OG) enzymes are found in all kingdoms of life and catalyze a broad array of oxidative transformations. Herein, we demonstrate that the activity of an Fe/2OG enzyme can be redirected when changing the targeted carbon hybridization from sp3 to sp2. During leucine 5-hydroxylase catalysis, installation of an olefin group onto the substrate redirects the Fe(IV)-oxo species reactivity from hydroxylation to asymmetric epoxidation. The resulting epoxide subsequently undergoes intramolecular cyclization to form the substituted piperidine, 2S,5S-hydroxypipecolic acid.}, number={21}, journal={BIOCHEMISTRY}, publisher={American Chemical Society (ACS)}, author={Cha, Lide and Milikisiyants, Sergey and Davidson, Madison and Xue, Shan and Smirnova, Tatyana I and Smirnov, Alex I and Guo, Yisong and Chang, Wei-Chen}, year={2020}, month={Jun}, pages={1961–1965} }
 @article{milikisiyants_voinov_marek_jafarabadi_liu_han_wang_smirnov_2019, title={Enhancing sensitivity of Double Electron-Electron Resonance (DEER) by using Relaxation-Optimized Acquisition Length Distribution (RELOAD) scheme}, volume={298}, ISSN={1090-7807}, url={http://dx.doi.org/10.1016/J.JMR.2018.12.004}, DOI={10.1016/j.jmr.2018.12.004}, abstractNote={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.}, journal={Journal of Magnetic Resonance}, publisher={Elsevier BV}, author={Milikisiyants, Sergey and Voinov, Maxim A. and Marek, Antonin and Jafarabadi, Morteza and Liu, Jing and Han, Rong and Wang, Shenlin and Smirnov, Alex I.}, year={2019}, month={Jan}, pages={115–126} }
 @article{yu_tang_cha_milikisiyants_smirnova_smirnov_guo_chang_2018, title={Elucidating the Reaction Pathway of Decarboxylation-Assisted Olefination Catalyzed by a Mononuclear Non-Heme Iron Enzyme}, volume={140}, ISSN={["1520-5126"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85056414780&partnerID=MN8TOARS}, DOI={10.1021/jacs.8b10077}, abstractNote={Installation of olefins into molecules is a key transformation in organic synthesis. The recently discovered decarboxylation-assisted olefination in the biosynthesis of rhabduscin by a mononuclear non-heme iron enzyme ( P.IsnB) represents a novel approach in olefin construction. This method is commonly employed in natural product biosynthesis. Herein, we demonstrate that a ferryl intermediate is used for C-H activation at the benzylic position of the substrate. We further establish that P.IsnB reactivity can be switched from olefination to hydroxylation using electron-withdrawing groups appended on the phenyl moiety of the analogues. These experimental observations imply that a pathway involving an initial C-H activation followed by a benzylic carbocation species or by electron transfer coupled β-scission is likely utilized to complete C═C bond formation.}, number={45}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Yu, Cheng-Ping and Tang, Yijie and Cha, Lide and Milikisiyants, Sergey and Smirnova, Tatyana I. and Smirnov, Alex I. and Guo, Yisong and Chang, Wei-chen}, year={2018}, month={Nov}, pages={15190–15193} }
 @article{nevzorov_milikisiyants_marek_smirnov_2018, title={Multi-resonant photonic band-gap/saddle coil DNP probehead for static solid state NMR of microliter volume samples}, volume={297}, ISSN={["1096-0856"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85055585775&partnerID=MN8TOARS}, DOI={10.1016/j.jmr.2018.10.010}, abstractNote={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.}, journal={JOURNAL OF MAGNETIC RESONANCE}, author={Nevzorov, Alexander A. and Milikisiyants, Sergey and Marek, Antonin N. and Smirnov, Alex I.}, year={2018}, month={Dec}, pages={113–123} }
 @article{milikisiyants_nevzorov_smirnov_2018, title={Photonic band-gap resonators for high-field/high-frequency EPR of microliter-volume liquid aqueous samples}, volume={296}, ISSN={["1096-0856"]}, url={https://doi.org/10.1016/j.jmr.2018.09.006}, DOI={10.1016/j.jmr.2018.09.006}, abstractNote={High-field EPR provides significant advantages for studying structure and dynamics of molecular systems possessing an unpaired electronic spin. However, routine use of high-field EPR in biophysical research, especially for aqueous biological samples, is still facing substantial technical difficulties stemming from high dielectric millimeter wave (mmW) losses associated with non-resonant absorption by water and other polar molecules. The strong absorbance of mmW’s by water also limits the penetration depth to just fractions of mm or even less, thus making fabrication of suitable sample containers rather challenging. Here we describe a radically new line of high Q-factor mmW resonators that are based on forming lattice defects in one-dimensional photonic band-gap (PBG) structures composed of low-loss ceramic discs of λ/4 in thickness and having alternating dielectric constants. A sample (either liquid or solid) is placed within the E = 0 node of the standing mm wave confined within the defect. A resonator prototype has been built and tested at 94.3 GHz. The resonator performance is enhanced by employing ceramic nanoporous membranes as flat sample holders of controllable thickness and tunable effective dielectric constant. The experimental Q-factor of an empty resonator was ≈ 420. The Q-factor decreased slightly to ≈ 370 when loaded with a water-containing nanoporous disc of 50 μm in thickness. The resonator has been tested with a number of liquid biological samples and demonstrated about tenfold gain in concentration sensitivity vs. a high-Q cylindrical TE012-type cavity. Detailed HFSS Ansys simulations have shown that the resonator structure could be further optimized by properly choosing the thickness of the aqueous sample and employing metallized surfaces. The PBG resonator design is readily scalable to higher mmW frequencies and is capable of accommodating significantly larger sample volumes than previously achieved with either Fabry-Perot or cylindrical resonators.}, journal={JOURNAL OF MAGNETIC RESONANCE}, publisher={Elsevier BV}, author={Milikisiyants, Sergey and Nevzorov, Alexander A. and Smirnov, Alex I}, year={2018}, month={Nov}, pages={152–164} }
 @article{shames_smirnov_milikisiyants_danilov_nunn_mcguire_torelli_shenderova_2017, title={Fluence-Dependent Evolution of Paramagnetic Triplet Centers in e-Beam Irradiated Microcrystalline lb Type HPHT Diamond}, volume={121}, ISSN={["1932-7447"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85031327261&partnerID=MN8TOARS}, DOI={10.1021/acs.jpcc.7b06514}, abstractNote={Paramagnetic triplet centers produced by e-beam irradiation of synthetic microcrystalline Ib-type high-pressure high-temperature (HPHT) diamonds were studied by continuous wave (CW) electron paramagnetic resonance (EPR) spectroscopy at X-band (9.4 GHz), pulsed EPR at X- and Q-bands (34 GHz), and fluorescence spectroscopies as a function of radiation fluences up to 5 × 1019 e–/cm2. EPR spectra of mostly “forbidden” Δms = 2 electronic spin transitions observed at g ≈ 4 (i.e., so-called half-field EPR spectra) reveal the presence of the main W15 triplet defects associated with the fluorescent negatively charged nitrogen-vacancy (NV–) centers as well as additional triplet spin centers identified as W16, W17, W18, and W33 that appear upon increasing the e-beam fluence. Consequent annealing at 1,400 °C significantly reduces the content of W17, W18, and W33 but not W15 and W16 defects. The efficacy of NV– center fabrication as a function of fluence dependent e-beam irradiation is also reported.}, number={40}, journal={JOURNAL OF PHYSICAL CHEMISTRY C}, author={Shames, Alexander I. and Smirnov, Alex I. and Milikisiyants, Sergey and Danilov, Evgeny O. and Nunn, Nicholas and McGuire, Gary and Torelli, Marco D. and Shenderova, Olga}, year={2017}, month={Oct}, pages={22335–22346} }
 @article{milikisiyants_wang_munro_donohue_ward_bolton_brown_smirnova_ladizhansky_smirnov_2017, title={Oligomeric Structure of Anabaena Sensory Rhodopsin in a Lipid Bilayer Environment by Combining Solid-State NMR and Long-range DEER Constraints}, volume={429}, ISSN={["1089-8638"]}, url={https://doi.org/10.1016/j.jmb.2017.05.005}, DOI={10.1016/j.jmb.2017.05.005}, abstractNote={Oligomerization of membrane proteins is common in nature. Here, we combine spin-labeling double electron-electron resonance (DEER) and solid-state NMR (ssNMR) spectroscopy to refine the structure of an oligomeric integral membrane protein, Anabaena sensory rhodopsin (ASR), reconstituted in a lipid environment. An essential feature of such a combined approach is that it provides structural distance restraints spanning a range of ca 3-60Å while using the same sample preparation (i.e., mutations, paramagnetic labeling, and reconstitution in lipid bilayers) for both ssNMR and DEER. Direct modeling of the multispin effects on DEER signal allowed for the determination of the oligomeric order and for obtaining long-range DEER distance restraints between the ASR trimer subunits that were used to refine the ssNMR structure of ASR. The improved structure of the ASR trimer revealed a more compact packing of helices and side chains at the intermonomer interface, compared to the structure determined using the ssNMR data alone. The extent of the refinement is significant when compared with typical helix movements observed for the active states of homologous proteins. Our combined approach of using complementary DEER and NMR measurements for the determination of oligomeric structures would be widely applicable to membrane proteins where paramagnetic tags can be introduced. Such a method could be used to study the effects of the lipid membrane composition on protein oligomerization and to observe structural changes in protein oligomers upon drug, substrate, and co-factor binding.}, number={12}, journal={JOURNAL OF MOLECULAR BIOLOGY}, publisher={Elsevier BV}, author={Milikisiyants, Sergey and Wang, Shenlin and Munro, Rachel A. and Donohue, Matthew and Ward, Meaghan E. and Bolton, David and Brown, Leonid S. and Smirnova, Tatyana I. and Ladizhansky, Vladimir and Smirnov, Alex I.}, year={2017}, month={Jun}, pages={1903–1920} }
 @article{voinov_good_ward_milikisiyants_marek_caporini_rosay_munro_ljumovic_brown_et al._2015, title={Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR}, volume={119}, ISSN={1520-6106 1520-5207}, url={http://dx.doi.org/10.1021/ACS.JPCB.5B05230}, DOI={10.1021/acs.jpcb.5b05230}, abstractNote={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.}, number={32}, journal={The Journal of Physical Chemistry B}, publisher={American Chemical Society (ACS)}, author={Voinov, Maxim A. and Good, Daryl B. and Ward, Meaghan E. and Milikisiyants, Sergey and Marek, Antonin and Caporini, Marc A. and Rosay, Melanie and Munro, Rachel A. and Ljumovic, Milena and Brown, Leonid S. and et al.}, year={2015}, month={Jul}, pages={10180–10190} }
 @article{marek_tang_milikisiyants_nevzorov_smirnov_2015, title={Nanotube Array Method for Studying Lipid-Induced Conformational Changes of a Membrane Protein by Solid-State NMR}, volume={108}, ISSN={["1542-0086"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84921058848&partnerID=MN8TOARS}, DOI={10.1016/j.bpj.2014.11.011}, abstractNote={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.}, number={1}, journal={BIOPHYSICAL JOURNAL}, publisher={Elsevier BV}, author={Marek, Antonin and Tang, Wenxing and Milikisiyants, Sergey and Nevzorov, Alexander A. and Smirnov, Alex I.}, year={2015}, month={Jan}, pages={5–9} }