@article{galiakhmetov_shah_lane_davern_proulx_nevzorov_2024, title={Peptoid-based macrodiscs of variable lipid composition for structural studies of membrane proteins by oriented-sample solid-state NMR}, volume={9}, ISSN={["2590-1524"]}, DOI={10.1016/j.yjsbx.2023.100095}, abstractNote={Solid-state Nuclear Magnetic Resonance (NMR) in combination with magnetically aligned discoidal lipid mimics allows for studying the conformations of membrane proteins in planar, lipid-rich bilayer environments and at the physiological temperature. We have recently demonstrated the general applicability of macrodiscs composed of DMPC lipids and peptoid belts, which yield magnetic alignment and NMR spectroscopic resolution comparable or superior to detergent-containing bicelles. Here we report on a considerable improvement in the magnetic alignment and NMR resolution of peptoid-based macrodiscs consisting of a mixture of the zwitterionic and negatively charged lipids (DMPC/DMPG at the 85% to 15% molar ratio). The resulting linewidths are about 30% sharper due to the higher orientational order parameter likely arising from the stabilizing electrostatic repulsion between the discs. Moreover, highly aligned, detergent-free macrodiscs can be formed with a longer-chain lipid, DPPC. Interestingly, the spectra of Pf1 in the two lipid mimetics are almost indistinguishable, which would mean that the overall transmembrane helix tilt might be governed not only by the hydrophobic matching but also possibly by the interactions of the flanking lysine and arginine residues at the membrane interface.}, journal={JOURNAL OF STRUCTURAL BIOLOGY-X}, author={Galiakhmetov, Azamat R. and Shah, Adit A. and Lane, Addison and Davern, Carolynn M. and Proulx, Caroline and Nevzorov, Alexander A.}, year={2024}, month={Jun} }
 @article{galiakhmetov_davern_esteves_awosanya_guthrie_proulx_nevzorov_2022, title={Aligned peptoid-based macrodiscs for structural studies of membrane proteins by oriented-sample NMR}, volume={121}, ISSN={["1542-0086"]}, DOI={10.1016/j.bpj.2022.07.024}, abstractNote={Development of a robust, uniform, and magnetically orientable lipid mimetic will undoubtedly advance solid-state NMR of macroscopically aligned membrane proteins. Here, we report on a novel lipid membrane mimetic based on peptoid belts. The peptoids, composed of 15 residues, were synthesized by alternating N-(2-phenethyl)glycine with N-(2-carboxyethyl)glycine residues at a 2:1 molar ratio. The chemically synthesized peptoids possess a much lower degree of polydispersity versus styrene-maleic acid polymers, thus yielding uniform discs. Moreover, the peptoid oligomers are more flexible and do not require a specific folding, unlike lipoproteins, in order to wrap around the hydrophobic membrane core. The NMR spectra measured for the membrane-bound form of Pf1 coat protein incorporated in this new lipid mimetics demonstrate a higher order parameter and uniform linewidths compared with the conventional bicelles and peptide-based macrodiscs. Importantly, unlike bicelles, the peptoid-based macrodiscs are detergent free.}, number={17}, journal={BIOPHYSICAL JOURNAL}, author={Galiakhmetov, Azamat R. and Davern, Carolynn M. and Esteves, Richard J. A. and Awosanya, Emmanuel O. and Guthrie, Quibria A. E. and Proulx, Caroline and Nevzorov, Alexander A.}, year={2022}, month={Sep}, pages={3263–3270} }
 @article{lapin_nevzorov_2022, title={Validation of protein backbone structures calculated from NMR angular restraints using Rosetta (vol 73, pg 229, 2019)}, ISSN={["1573-5001"]}, DOI={10.1007/s10858-022-00398-w}, journal={JOURNAL OF BIOMOLECULAR NMR}, author={Lapin, Joel and Nevzorov, Alexander A.}, 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{lapin_awosanya_esteves_nevzorov_2021, title={H-1/C-13/N-15 triple-resonance experiments for structure determinaton of membrane proteins by oriented-sample NMR}, volume={111}, ISSN={["1527-3326"]}, DOI={10.1016/j.ssnmr.2020.101701}, abstractNote={The benefits of triple-resonance experiments for structure determination of macroscopically oriented membrane proteins by solid-state NMR are discussed. While double-resonance 1H/15N experiments are effective for structure elucidation of alpha-helical domains, extension of the method of oriented samples to more complex topologies and assessing side-chain conformations necessitates further development of triple-resonance (1H/13C/15N) NMR pulse sequences. Incorporating additional spectroscopic dimensions involving 13C spin-bearing nuclei, however, introduces essential complications arising from the wide frequency range of the 1H-13C dipolar couplings and 13C CSA (>20 ​kHz), and the presence of the 13C-13C homonuclear dipole-dipole interactions. The recently reported ROULETTE-CAHA pulse sequence, in combination with the selective z-filtering, can be used to evolve the structurally informative 1H-13C dipolar coupling arising from the aliphatic carbons while suppressing the signals from the carbonyl and methyl regions. Proton-mediated magnetization transfer under mismatched Hartman-Hahn conditions (MMHH) can be used to correlate 13C and 15N nuclei in such triple-resonance experiments for the subsequent 15N detection. The recently developed pulse sequences are illustrated for n-acetyl Leucine (NAL) single crystal and doubly labeled Pf1 coat protein reconstituted in magnetically aligned bicelles. An interesting observation is that in the case of 15N-labeled NAL measured at 13C natural abundance, the triple (1H/13C/15N) MMHH scheme predominantly gives rise to long-range intermolecular magnetization transfers from 13C to 15N spins; whereas direct Hartmann-Hahn 13C/15N transfer is entirely intramolecular. The presented developments advance NMR of oriented samples for structure determination of membrane proteins and liquid crystals.}, journal={SOLID STATE NUCLEAR MAGNETIC RESONANCE}, author={Lapin, Joel and Awosanya, Emmanuel O. and Esteves, Richard J. A. and Nevzorov, Alexander A.}, year={2021}, month={Feb} }
 @article{lapin_nevzorov_2020, title={Computer-generated pulse sequences for H-1-N-15 and H-1(alpha)-C-13(alpha) separated local-field experiments}, volume={317}, ISSN={["1096-0856"]}, DOI={10.1016/j.jmr.2020.106794}, abstractNote={High-resolution separated local field (SLF) experiments are employed in oriented-sample solid state NMR to measure angular-dependent heteronuclear dipolar couplings for structure determination. While traditionally these experiments have been designed analytically by determining cycles of pulses with specific phases and durations to achieve cancellation of the homonuclear dipolar terms in the average Hamiltonian, recent work has introduced a computational approach to optimizing linewidths of the 1H-15N dipolar resonances. Accelerated by GPU processors, a computer algorithm searches for the optimal parameters by simulating numerous 1H-15N NMR spectra. This approach, termed ROULETTE, showed promising results by developing a new pulse sequence (ROULETTE-1.0) exhibiting 18% sharper mean linewidths than SAMPI4 for an N-acetyl Leucine (NAL) crystal. Herein, we expand on this previous work to improve the performance of the 1H-15N SLF experiment and extend the work beyond the original approach to new SLF experiments. The new algorithm, in addition to finding pulse durations and phases, now searches for the optimal on/off application scheme of radio frequency irradiation on each channel. This constitutes true de novo optimization, effectively optimizing every aspect of a pulse sequence instead of just phases and durations. With an improved ROULETTE algorithm, we have found a new 1H-15N pulse sequence, termed ROULETTE-2.0, yielding 32% sharper mean linewidths than SAMPI4 for NAL crystal at 500 MHz 1H frequency. Whereas both SAMPI4 and ROULETTE-1.0 have a window where the rf power on the I-channel is turned off, the new pulse sequence is entirely windowless. Furthermore, the reliability of the algorithm has been greatly improved in terms of avoiding false positives, i.e. well-performing pulse sequences in silica that fail to render narrow resonances in experiment. The program has been extended to the 13Cα-1Hα SLF experiments, using a 6 subdwell architecture similar to the 1H-15N optimization. Compared to the PISEMA pulse sequence, the mean 13Cα-1Hα linewidth is 17% sharper for the new pulse sequence, termed ROULETTE-CAHA. In addition to superior performance, the work demonstrates the broad applicability of the algorithm and its adaptability to different NMR experiments and spin systems.}, journal={JOURNAL OF MAGNETIC RESONANCE}, author={Lapin, Joel and Nevzorov, Alexander A.}, year={2020}, month={Aug} }
 @article{lapin_nevzorov_2020, title={De novo NMR pulse sequence design using Monte-Carlo optimization techniques}, volume={310}, ISBN={1096-0856}, DOI={10.1016/j.jmr.2019.106641}, abstractNote={Separated Local Field (SLF) experiments have been routinely used for measuring 1H-15N heteronuclear dipolar couplings in oriented-sample solid-state NMR for structure determination of proteins. In the on-going pursuit of designing better-performing SLF pulse sequences (e.g. by increasing the number of subdwells, and varying the rf amplitudes and phases), analytical treatment of the relevant average Hamiltonian terms may become cumbersome and/or nearly impossible. Numerical simulations of NMR experiments using GPU processors can be employed to rapidly calculate spectra for moderately sized spin systems, which permit an efficient numeric optimization of pulse sequences by the Monte Carlo Simulated Annealing protocol. In this work, a computational strategy was developed to find the optimal phases and timings that substantially improve the 1H-15N dipolar linewidths over a broad range of dipolar couplings as compared to SAMPI4. More than 100 pulse sequences were developed de novo and tested on an N-acetyl Leucine crystal. Seventeen distinct pulse sequences were shown to produce sharper mean linewidths than SAMPI4. Overall, these pulse sequences have more variable parameters (involving non-quadrature phases) and do not involve symmetry between the odd and even dwells, which would likely preclude their rigorous analytical treatment. The top performing pulse sequence, termed ROULETTE-1, has 18% sharper mean linewidths than SAMPI4 when run on an N-acetyl Leucine crystal. This sequence was also shown to be robust over a broad range of 1H carrier frequencies and various crystal orientations. The performance of such an optimized pulse sequence was also illustrated on 15N Leucine-labeled Pf1 coat protein reconstituted in magnetically aligned bicelles. For the optimized pulse sequence the mean peak width was 14% sharper than SAMPI4, which in turn yielded a better signal to noise ratio, 20:1 vs. 17:1. This method is potentially extendable to de novo development of a variety of NMR experiments.}, journal={JOURNAL OF MAGNETIC RESONANCE}, author={Lapin, Joel and Nevzorov, Alexander A.}, year={2020}, month={Jan} }
 @article{awosanya_lapin_nevzorov_2020, title={NMR "Crystallography" for Uniformly (C-13, N-15)-Labeled Oriented Membrane Proteins}, volume={59}, ISSN={["1521-3773"]}, DOI={10.1002/anie.201915110}, abstractNote={Abstract}, number={9}, journal={ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, author={Awosanya, Emmanuel O. and Lapin, Joel and Nevzorov, Alexander A.}, year={2020}, month={Feb}, pages={3554–3557} }
 @article{jafarabadi_chestnut_marek_nevzorov_smirnov_2019, title={Nesting Lipid Bilayers in Nanopores: Effect of Pore Diameter on Macroscopic Order and the Layer Count}, volume={116}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2018.11.478}, DOI={10.1016/J.BPJ.2018.11.478}, abstractNote={Nanotubular lipid bilayer membranes formed by self-assembly and confined by macroscopically aligned nanopores exhibit several attractive features for biophysics and bio-nanotechnology. When fully hydrated such bilayers systems are stable for at least a month and allow for a quick exchange of buffers and other water-soluble molecules. Even more importantly the bilayers have well-defined curvature and are macroscopically aligned over an exceptionally broad range of temperature, pH, and ionic strength. The latter feature facilitates studies of membrane and membrane proteins under close to native cellular membrane conditions by solid-state NMR and other spectroscopic methods. Here we report on the effect of nanoscale pore diameter on packing of lipid bilayers and macroscopic alignment of POPC (2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine) lipids inside the pores. We show that the pores from 36 to 44, 50 and 72 nm in diameter accommodate progressively larger number of nesting lipid bilayers – from up to two nanotubes for the smallest 36 nm pore to about four for 50 nm wide pore at 40 oC. This is consistent with 26-27 nm being the smallest diameter for the POPC nanotubes. Static 31P linewidth at 300 MHz 1H frequency indicated the best macroscopic alignment corresponding to just 1-3% of mosaic spread for either one or two nesting lipid nanotubes for all pore sizes. 31P linewidth and bilayer macroscopic alignment degraded notably for three nesting bilayers formed in the nanopores and even more for four. The latter is consistent with a formation of wavy lipid tubules previously reported for much larger 140-180 nm nanopores. Supported by DE-FG02-02ER15354 to AIS.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Jafarabadi, Morteza and Chestnut, Melanie and Marek, Antonin and Nevzorov, Alexander and Smirnov, Alex I.}, year={2019}, month={Feb}, pages={80a–81a} }
 @article{lapin_nevzorov_2019, title={Validation of protein backbone structures calculated from NMR angular restraints using Rosetta}, volume={73}, ISSN={["1573-5001"]}, DOI={10.1007/s10858-019-00251-7}, abstractNote={Multidimensional solid-state NMR spectra of oriented membrane proteins can be used to infer the backbone torsion angles and hence the overall protein fold by measuring dipolar couplings and chemical shift anisotropies, which depend on the orientation of each peptide plane with respect to the external magnetic field. However, multiple peptide plane orientations can be consistent with a given set of angular restraints. This ambiguity is further exacerbated by experimental uncertainty in obtaining and interpreting such restraints. The previously developed algorithms for structure calculations using angular restraints typically involve a sequential walkthrough along the backbone to find the torsion angles between the consecutive peptide plane orientations that are consistent with the experimental data. This method is sensitive to experimental uncertainty in interpreting the peak positions of as low as ± 10 Hz, often yielding high structural RMSDs for the calculated structures. Here we present a significantly improved version of the algorithm which includes the fitting of several peptide planes at once in order to prevent propagation of error along the backbone. In addition, a protocol has been devised for filtering the structural solutions using Rosetta scoring functions in order to find the structures that both fit the spectrum and satisfy bioinformatics restraints. The robustness of the new algorithm has been tested using synthetic angular restraints generated from the known structures for two proteins: a soluble protein 2gb1 (56 residues), chosen for its diverse secondary structure elements, i.e. an alpha-helix and two beta-sheets, and a membrane protein 4a2n, from which the first two transmembrane helices (having a total of 64 residues) have been used. Extensive simulations have been performed by varying the number of fitted planes, experimental error, and the number of NMR dimensions. It has been found that simultaneously fitting two peptide planes always shifted the distribution of the calculated structures toward lower structural RMSD values as compared to fitting a single torsion-angle pair. For each protein, irrespective of the simulation parameters, Rosetta was able to distinguish the most plausible structures, often having structural RMSDs lower than 2 Å with respect to the original structure. This study establishes a framework for de-novo protein structure prediction using a combination of solid-state NMR angular restraints and bioinformatics.}, number={5}, journal={JOURNAL OF BIOMOLECULAR NMR}, author={Lapin, Joel and Nevzorov, Alexander A.}, year={2019}, month={May}, pages={229–244} }
 @article{lapin_nevzorov_2018, title={Automated assignment of NMR spectra of macroscopically oriented proteins using simulated annealing}, volume={293}, ISSN={1090-7807}, url={http://dx.doi.org/10.1016/J.JMR.2018.06.004}, DOI={10.1016/J.JMR.2018.06.004}, abstractNote={An automated technique for the sequential assignment of NMR backbone resonances of oriented protein samples has been developed and tested based on 15N-15N homonuclear exchange and spin-exchanged separated local-field spectra. By treating the experimental spectral intensity as a pseudopotential, the Monte-Carlo Simulated Annealing algorithm has been employed to seek lowest-energy assignment solutions over a large sampling space where direct enumeration would be unfeasible. The determined sequential assignments have been scored based on the positions of the crosspeaks resulting from the possible orders for the main peaks. This approach is versatile in terms of the parameters that can be specified to achieve the best-fit result. At a minimum the algorithm requires a continuous segment of the main-peak chemical shifts obtained from a uniformly labeled sample and a spin-exchanged experimental spectrum represented as a 2D matrix array. With selective labeling experiments, groups of chemical shifts corresponding to specific locations in the protein backbone can be fixed, thereby decreasing the sampling space. The output from the program consists of a list of top-score main peak assignments, which can be subjected to further scoring criteria until a consensus solution is found. The algorithm has first been tested on a synthetic spectrum with randomly generated chemical shifts and dipolar couplings for the main peaks. The original assignments have been successfully recovered for as many as 100 main peaks when residue-type information was used even in the presence of substantial spectral peak overlap. The algorithm was then applied to assigning two sets of experimental spectra to recover and confirm the previously established assignments in an automated fashion. For the 20-residue transmembrane domain of Pf1 coat protein reconstituted in magnetically aligned bicelles, the original assignment by Park et al. (2010) was recovered by the automated algorithm with additional input from 5 selectively labeled amino acid spectra. The second case considered was the 46 residue Pf1 bacteriophage from Thiriot et al. (2005) and Knox et al. (2010), of which 38 residues were fit. Automated fitting resulted in several possible assignments but not exactly the original assignment. By using a post-fitting filtering procedure based on the number of missed cross peaks and Pf1 helical structure, a consensus spectroscopic assignment is proposed covering 84% of the original assignment. While the automated assignment works best in spectra with well-resolved crosspeaks, it also tolerates substantial spectral crowding to yield reasonable assignments in the cases where ambiguity and degeneracy of possible assignment solutions are inevitable.}, journal={Journal of Magnetic Resonance}, publisher={Elsevier BV}, author={Lapin, Joel and Nevzorov, Alexander A.}, year={2018}, month={Aug}, pages={104–114} }
 @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{awosanya_nevzorov_2018, title={Protein Rotational Dynamics in Aligned Lipid Membranes Probed by Anisotropic T-1 rho NMR Relaxation}, volume={114}, ISSN={["1542-0086"]}, DOI={10.1016/j.bpj.2017.11.3740}, abstractNote={A membrane-bound form of Pf1 coat protein reconstituted in magnetically aligned DMPC/DHPC bicelles was used as a molecular probe to quantify for the viscosity of the lipid membrane interior by measuring the uniaxial rotational diffusion coefficient of the protein. Orientationally dependent 15N NMR relaxation times in the rotating frame, or T1ρ, were determined by fitting individually the decay of the resolved NMR peaks corresponding to the transmembrane helix of Pf1 coat protein as a function of the spin-lock time incorporated into the 2D SAMPI4 pulse sequence. The T1ρ relaxation mechanism was modeled by uniaxial rotational diffusion on a cone, which yields a linear correlation with respect to the bond factor sin4θB, where θB is the angle that the NH bond forms with respect to the axis of rotation. Importantly, the bond factors can be independently measured from the dipolar couplings in the separated local-field SAMPI4 spectra. From this dependence, the value of the diffusion coefficient D|| = 8.0 × 105 s−1 was inferred from linear regression of the experimental T1ρ data even without any spectroscopic assignment. Alternatively, a close value of D|| = 7.7 × 105 s−1 was obtained by fitting the T1ρ relaxation data for the assigned NMR peaks of the transmembrane domain of Pf1 to a wavelike pattern as a function of residue number. The method illustrates the use of single-helix transmembrane peptides as molecular probes to assess the dynamic parameters of biological membranes by NMR relaxation in oriented lipid bilayers.}, number={2}, journal={BIOPHYSICAL JOURNAL}, author={Awosanya, Emmanuel O. and Nevzorov, Alexander A.}, year={2018}, month={Jan}, pages={392–399} }
 @article{nevzorov_awosanya_2018, title={Rotational Diffusion of Membrane Proteins Probed by Anisotropic T2 and T1ρ NMR Relaxation in Aligned Lipid Bilayers}, volume={114}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2017.11.1343}, DOI={10.1016/J.BPJ.2017.11.1343}, abstractNote={Membrane proteins are known to undergo fast uniaxial rotational diffusion about the membrane normal on the microsecond scale. Accurate determination of local bilayer viscosity can provide quantitative information about the environment surrounding membrane-embedded proteins. Using Pf1 coat protein reconstituted in aligned DMPC/DHPC bicelles as a probe, rotational diffusion coefficients and membrane viscosity were assessed. The diffusion coefficients were independently measured by T2 and T1ρ relaxation rates, which depend on the heteronuclear dipolar couplings. A model for interpreting anisotropic relaxation was developed based on uniaxial diffusion on a cone. The 15N T1ρ relaxation rates have been determined by fitting individually the decay of NMR peaks for the Pf1 transmembrane domain using a modified SAMPI4 sequence. The model yields a linear correlation with respect to the bond factor sin4θB, where the NH bond angle θB can be independently measured from the SAMPI4 spectrum. The diffusion coefficient D|| = 8.0×105 s−1 was determined from the linear regression of the experimental T1ρ data even without any spectroscopic assignment. Remarkably, the assigned R1ρ relaxation data for the helical domain of Pf1 can be fitted to a wave-like pattern having a periodicity of 3.6, which yields a close value of D|| = 7.7×105 s−1. A reasonably close value (D|| = 5×105 s−1) has been obtained from the fitting of T2 relaxation data, though the latter has additional contributions arising from magnetic field inhomogeneity, incomplete dipolar decoupling, and inhomogeneous contribution from the sample mosaicity. The experimentally obtained diffusion coefficients are consistent with simple estimates using the Stokes-Einstein equation if the protein is modeled as a rigid cylinder having the radius of 10 Å and local bilayer viscosity of 1 Poise, i.e. similar to the classical value of Saffman and Delbruck.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Nevzorov, Alexander and Awosanya, Emmanuel}, year={2018}, month={Feb}, pages={241a} }
 @article{tesch_pourmoazzen_awosanya_nevzorov_2018, title={Uniaxial Diffusional Narrowing of NMR Lineshapes for Membrane Proteins Reconstituted in Magnetically Aligned Bicelles and Macrodiscs}, volume={49}, ISSN={["1613-7507"]}, DOI={10.1007/s00723-018-1056-4}, number={12}, journal={APPLIED MAGNETIC RESONANCE}, author={Tesch, Deanna M. and Pourmoazzen, Zhaleh and Awosanya, Emmanuel O. and Nevzorov, Alexander A.}, year={2018}, month={Dec}, pages={1335–1353} }
 @article{koroloff_nevzorov_2017, title={Selective excitation for spectral editing and assignment in separated local field experiments of oriented membrane proteins}, volume={274}, ISSN={1090-7807}, url={http://dx.doi.org/10.1016/J.JMR.2016.10.013}, DOI={10.1016/J.JMR.2016.10.013}, abstractNote={Spectroscopic assignment of NMR spectra for oriented uniformly labeled membrane proteins embedded in their native-like bilayer environment is essential for their structure determination. However, sequence-specific assignment in oriented-sample (OS) NMR is often complicated by insufficient resolution and spectral crowding. Therefore, the assignment process is usually done by a laborious and expensive "shotgun" method involving multiple selective labeling of amino acid residues. Presented here is a strategy to overcome poor spectral resolution in crowded regions of 2D spectra by selecting resolved "seed" residues via soft Gaussian pulses inserted into spin-exchange separated local-field experiments. The Gaussian pulse places the selected polarization along the z-axis while dephasing the other signals before the evolution of the 1H-15N dipolar couplings. The transfer of magnetization is accomplished via mismatched Hartmann-Hahn conditions to the nearest-neighbor peaks via the proton bath. By optimizing the length and amplitude of the Gaussian pulse, one can also achieve a phase inversion of the closest peaks, thus providing an additional phase contrast. From the superposition of the selective spin-exchanged SAMPI4 onto the fully excited SAMPI4 spectrum, the 15N sites that are directly adjacent to the selectively excited residues can be easily identified, thereby providing a straightforward method for initiating the assignment process in oriented membrane proteins.}, journal={Journal of Magnetic Resonance}, publisher={Elsevier BV}, author={Koroloff, Sophie N. and Nevzorov, Alexander A.}, year={2017}, month={Jan}, pages={7–12} }
 @article{koroloff_tesch_awosanya_nevzorov_2017, title={Sensitivity enhancement for membrane proteins reconstituted in parallel and perpendicular oriented bicelles obtained by using repetitive cross-polarization and membrane-incorporated free radicals}, volume={67}, ISSN={["1573-5001"]}, DOI={10.1007/s10858-017-0090-0}, abstractNote={Multidimensional separated local-field and spin-exchange experiments employed by oriented-sample solid-state NMR are essential for structure determination and spectroscopic assignment of membrane proteins reconstituted in macroscopically aligned lipid bilayers. However, these experiments typically require a large number of scans in order to establish interspin correlations. Here we have shown that a combination of optimized repetitive cross polarization (REP-CP) and membrane-embedded free radicals allows one to enhance the signal-to-noise ratio by factors 2.4-3.0 in the case of Pf1 coat protein reconstituted in magnetically aligned bicelles with their normals being either parallel or perpendicular to the main magnetic field. Notably, spectral resolution is not affected at the 2:1 radical-to-protein ratio. Spectroscopic assignment of Pf1 coat protein in the parallel bicelles has been established as an illustration of the method. The proposed methodology will advance applications of oriented-sample NMR technique when applied to samples containing smaller quantities of proteins and three-dimensional experiments.}, number={2}, journal={JOURNAL OF BIOMOLECULAR NMR}, author={Koroloff, Sophie N. and Tesch, Deanna M. and Awosanya, Emmanuel O. and Nevzorov, Alexander A.}, year={2017}, month={Feb}, pages={135–144} }
 @article{nevzorov_jafarabadi_marek_smirnov_2017, title={Structure and Dynamics of Nanopore-Confined Membrane Proteins are Affected by Bilayer Lipid Composition}, volume={112}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2016.11.2109}, DOI={10.1016/J.BPJ.2016.11.2109}, abstractNote={Self-assembled nanotubular lipid bilayers confined within macroscopically aligned high-density homogeneous nanopore array structure of anodic aluminum oxide (AAO) membranes exhibit a high degree of macroscopic alignment that facilitates their studies by oriented sample solid state (OS SS) NMR and spin-labeling EPR methods. The principal advantages of the lipid nanotube arrays are in the applicability of this method to essentially any lipid composition and a tolerance of the macroscopic alignment to a wide range of environmental conditions such as, e.g., temperature, pH, and ionic strength. Here we describe 1) a recent progress in reconstructing small pore-forming and transmembrane peptides as well as large membrane protein complexes, such as photosynthetic reaction center (RC) protein in inorganic nanopores and 2) utilization of this technology to study lipid-induced changes in protein conformations by magnetic resonance methods. Specifically, by using OS SS NMR we have shown that the tilt and α-helix kink angles of Pf1 coat protein are affected by the bilayer composition on example of DOPC, POPC and DMPC bilayers. Effects of unsaturated lipids on Pf1 dynamics as evidenced by the changes in the linewidths in the Pf1 spectra are also reported. We relate these changes to the bilayer fluidity and its effects on the uniaxial rotational diffusion of the protein within the membrane. Further, we demonstrate the use of DEER spectroscopy to obtain long-range distance constraints for membrane protein systems incorporated into lipid nanotubes. The improved nanopore alignment technique described here provides a general method for studying lipid-induced structural conformations of membrane proteins under physiologically relevant conditions by magnetic resonance. Supported by U.S. DOE contract No. DE-FG02-02ER15354 to A.I.S. and NSF MRI 1229547 to A.A.N.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Nevzorov, Alexander A. and Jafarabadi, Morteza and Marek, Antonin and Smirnov, Alex I.}, year={2017}, month={Feb}, pages={388a} }
 @article{jafarabadi_marek_koolivand_acharya_nevzorov_krim_smirnov_2016, title={Interactions of Antibacterial Peptides with Nanotubular Lipid Bilayers: Binding Kinetics and Distortions of the Bilayer Structure}, volume={110}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2015.11.488}, DOI={10.1016/J.BPJ.2015.11.488}, abstractNote={Interactions of peptides with membranes are central for understanding key cellular processes involving membrane proteins such as folding, signaling, transport, energy conversion, and immune response. These interactions also determine efficiency of antibacterial and cytotoxic peptides in disrupting the barrier function of cellular membranes. We employed macroscopically aligned tubular lipid bilayers confined inside cylindrical nanopores of anodic aluminum oxide (AAO) as a versatile nanotechnology platform to study membrane interactions of antibacterial peptides of melittin and alamecitin. Kinetics of peptide binding to lipid nanotubes and the eventual lipid removal/lysis (for melittin) were observed by quartz crystal microbalance (QCM) using a crystal with an in-house fabricated nanostructured surface while the structural changes in bilayers were monitored by solid state oriented sample NMR. Specifically, using 78 nm nanopores we have achieved exceptionally narrow (100-140 Hz or <1 ppm) 31P resonances of the lipid phosphate groups of macroscopically aligned nanotubular bilayers that indicated <1-2o mosaic spread in the lipid alignment in the absence of bioreactive peptides. 31P resonances broadened and shifted towards the isotropic values upon interacting with melittin with some lysis of lipids observed after several hours of exposure. The binding and lipid lysis from bilayers confined in essentially the same nanopores albeit at much smaller lipid quantity were further quantified by real-time QCM for bilayers of various lipid compositions. Taken together, the data relate peptide binding and lysis kinetics to structural changes in lipid bilayers, thus, shedding the light on the underlying multistep mechanism. The main advantage of the lipid nanotube AAO platform is in its versatile applicability to various biophysical methods such QCM and NMR under essentially the same environmental conditions such as pH, ionic strength, temperature, etc. and exceptionally broad range of lipid bilayer compositions.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Jafarabadi, Morteza and Marek, Antonin and Koolivand, Amir and Acharya, Biplav and Nevzorov, Alexander A. and Krim, Jacqueline and Smirnov, Alex I.}, year={2016}, month={Feb}, pages={79a} }
 @article{nevzorov_smirnov_2015, title={Line narrowing in oriented-sample NMR of membrane proteins}, volume={32}, journal={Protein nmr: modern techniques and biomedical applications}, author={Nevzorov, A. A. and Smirnov, A. I.}, year={2015}, pages={159–185} }
 @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} }
 @article{koroloff_nevzorov_2015, title={Optimization of cross-polarization at low radiofrequency fields for sensitivity enhancement in solid-state NMR of membrane proteins reconstituted in magnetically aligned bicelles}, volume={256}, ISSN={["1096-0856"]}, DOI={10.1016/j.jmr.2015.03.016}, abstractNote={Solid-state NMR (ssNMR) of oriented membrane proteins (MPs) is capable of providing structural and dynamic information at nearly physiological conditions. However, NMR experiments performed on oriented membrane proteins generally suffer from low sensitivity. Moreover, utilization of high-power radiofrequency (RF) irradiations for magnetization transfer may give rise to sample heating, thereby decreasing the efficiency of conventional cross-polarization schemes. Here we have optimized the recently developed repetitive cross-polarization (REP-CP) sequence (Tang et al., 2011) to further increase the magnetization transfer efficiency for membrane proteins reconstituted in magnetically aligned bicelles and compared its performance to single-contact Hartmann-Hahn cross-polarization (CP), CP-MOIST and the adiabatic transfer. It has been found that employing the REP-CP sequence at RF amplitudes of 19kHz instead of the commonly used higher RF fields (>45kHz) enhances the efficiency of REP-CP. An additional 30% signal can be obtained as compared to the previously published REP-CP, and 20% when compared to the re-optimized REP-CP at 50kHz RF fields. Moreover, the (15)N signal gain of low-power REP-CP was found to be 40% over the adiabatic CP and up to 80% over CP-MOIST. Thus, the low-power REP-CP sequence surpasses all of the previous CP schemes in addition of having the tremendous advantage of reducing the RF powers by a factor of seven, thereby preserving the liquid-like bicelle sample. By contrast, in purely static (NAL crystal) and semi-rigid systems (Pf1 phage), the adiabatic CP was found to be more effective. Periodic oscillations of the intensity profile (distinct from the transient oscillations) as a function of the CP contact time and B1 RF field strengths were observed during the REP-CP optimization with the oscillations becoming more pronounced with lower RF fields. Many-spin simulations were performed to explain the oscillations and their periodicity.}, journal={JOURNAL OF MAGNETIC RESONANCE}, author={Koroloff, Sophie N. and Nevzorov, Alexander A.}, year={2015}, month={Jul}, pages={14–22} }
 @article{nevzorov_2014, title={Coherent and stochastic averaging in solid-state NMR}, volume={249}, ISSN={["1096-0856"]}, DOI={10.1016/j.jmr.2014.09.023}, abstractNote={A new approach for calculating solid-state NMR lineshapes of uniaxially rotating membrane proteins under the magic-angle spinning conditions is presented. The use of stochastic Liouville equation (SLE) allows one to account for both coherent sample rotation and stochastic motional averaging of the spherical dipolar powder patterns by uniaxial diffusion of the spin-bearing molecules. The method is illustrated via simulations of the dipolar powder patterns of rigid samples under the MAS conditions, as well as the recent method of rotational alignment in the presence of both MAS and rotational diffusion under the conditions of dipolar recoupling. It has been found that it is computationally more advantageous to employ direct integration over a spherical grid rather than to use a full angular basis set for the SLE solution. Accuracy estimates for the bond angles measured from the recoupled amide 1H-15N dipolar powder patterns have been obtained at various rotational diffusion coefficients. It has been shown that the rotational alignment method is applicable to membrane proteins approximated as cylinders with radii of approximately 20Å, for which uniaxial rotational diffusion within the bilayer is sufficiently fast and exceeds the rate 2×105s-1.}, journal={JOURNAL OF MAGNETIC RESONANCE}, author={Nevzorov, Alexander A.}, year={2014}, month={Dec}, pages={9–15} }
 @article{peresin_vesterinen_habibi_johansson_pawlak_nevzorov_rojas_2014, title={Crosslinked PVA nanofibers reinforced with cellulose nanocrystals: Water interactions and thermomechanical properties}, volume={131}, ISSN={0021-8995}, url={http://dx.doi.org/10.1002/APP.40334}, DOI={10.1002/APP.40334}, abstractNote={ABSTRACT}, number={11}, journal={Journal of Applied Polymer Science}, publisher={Wiley}, author={Peresin, Maria Soledad and Vesterinen, Arja-Helena and Habibi, Youssef and Johansson, Leena-Sisko and Pawlak, Joel J. and Nevzorov, Alexander A. and Rojas, Orlando J.}, year={2014}, month={Jan}, pages={n/a-n/a} }
 @article{nevzorov_tesch_2014, title={Dynamic and Contrasting Information by Oriented-Sample Solid-State NMR Spectroscopy of Membrane Proteins}, volume={106}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/J.BPJ.2013.11.1138}, DOI={10.1016/J.BPJ.2013.11.1138}, abstractNote={Oriented-sample NMR (OS NMR) has emerged as a powerful technique for structure determination of membrane proteins in their native-like lipid environment. We have developed a model relating OS NMR lineshapes to uniaxial ordering (mosaic spread) and rotational diffusion of the protein within the membrane. The model is exemplified by 15N NMR spectra of Pf1 coat protein in both magnetically aligned phage and reconstituted in oriented bicelles. In the case of Pf1 phage, the lineshapes are dominated by static uniaxial disorder; whereas fast rotational diffusion of the protein is responsible for the motional line narrowing in perpendicularly oriented bicelles. From the analysis of 15N NMR linewidths, rotational diffusion coefficient can be estimated. Since the value of the diffusion coefficient is ultimately related to the overall protein size, information about oligomerization states is potentially obtainable. Second, the use of various membrane-embedded radicals allows one to both dramatically speed up data acquisition, on the one hand, and obtain contrasting information for membrane-embedded proteins, on the other. While membrane-bound paramagnetic species drastically affect the T1Z relaxation times (at 2:1 molar ratio relative to the protein), the transverse T2 relaxation is only slightly affected. 5-DOXYL stearic acid, TEMPOL, and CAT-1 radicals exhibit different partitioning within the membrane, and result in differential paramagnetic effect on the spectral peaks arising from different residues of Pf1 protein in bicelles. This allows one to obtain contrasting information about the location of the residues relative to the membrane. As was shown by EPR, TEMPOL partitions itself equally in and out of the membrane, and almost uniformly affects all residues within the bilayer. By contrast, 5-DOXYL stearic acid affects mostly the residues below the interfacial region, while CAT-1 affects the residues located within the polar head groups.}, number={2}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Nevzorov, Alexander and Tesch, Deanna M.}, year={2014}, month={Jan}, pages={192a} }
 @article{tesch_nevzorov_2014, title={Sensitivity enhancement and contrasting information provided by free radicals in oriented-sample NMR of bicelle-reconstituted membrane proteins}, volume={239}, ISSN={["1096-0856"]}, DOI={10.1016/j.jmr.2013.11.010}, abstractNote={Elucidating structure and topology of membrane proteins (MPs) is essential for unveiling functionality of these important biological constituents. Oriented-sample solid-state NMR (OS-NMR) is capable of providing such information on MPs under nearly physiological conditions. However, two dimensional OS-NMR experiments can take several days to complete due to long longitudinal relaxation times combined with the large number of scans to achieve sufficient signal sensitivity in biological samples. Here, free radicals 5-DOXYL stearic acid, TEMPOL, and CAT-1 were added to uniformly 15N-labeled Pf1 coat protein reconstituted in DMPC/DHPC bicelles, and their effect on the longitudinal relaxation times (T1Z) was investigated. The dramatically shortened T1Z’s allowed for the signal gain per unit time to be used for either: (i) up to a threefold reduction of the total experimental time at 99% magnetization recovery or (ii) obtaining up to 74% signal enhancement between the control and radical samples during constant experimental time at “optimal” relaxation delays. In addition, through OS-NMR and high-field EPR studies, free radicals were able to provide positional constraints in the bicelle system, which provide a description of the location of each residue in Pf1 coat protein within the bicellar membranes. This information can be useful in the determination of oligomerization states and immersion depths of larger membrane proteins.}, journal={JOURNAL OF MAGNETIC RESONANCE}, author={Tesch, Deanna M. and Nevzorov, Alexander A.}, year={2014}, month={Feb}, pages={9–15} }
 @article{edwards_savostyanov_nevzorov_concistre_pileio_kuprov_2013, title={Grid-free powder averages: On the applications of the Fokker-Planck equation to solid state NMR}, volume={235}, ISSN={["1096-0856"]}, DOI={10.1016/j.jmr.2013.07.011}, abstractNote={We demonstrate that Fokker–Planck equations in which spatial coordinates are treated on the same conceptual level as spin coordinates yield a convenient formalism for treating magic angle spinning NMR experiments. In particular, time dependence disappears from the background Hamiltonian (sample spinning is treated as an interaction), spherical quadrature grids are avoided completely (coordinate distributions are a part of the formalism) and relaxation theory with any linear diffusion operator is easily adopted from the Stochastic Liouville Equation theory. The proposed formalism contains Floquet theory as a special case. The elimination of the spherical averaging grid comes at the cost of increased matrix dimensions, but we show that this can be mitigated by the use of state space restriction and tensor train techniques. It is also demonstrated that low correlation order basis sets apparently give accurate answers in powder-averaged MAS simulations, meaning that polynomially scaling simulation algorithms do exist for a large class of solid state NMR experiments.}, journal={JOURNAL OF MAGNETIC RESONANCE}, author={Edwards, Luke J. and Savostyanov, D. V. and Nevzorov, A. A. and Concistre, M. and Pileio, G. and Kuprov, Ilya}, year={2013}, month={Oct}, pages={121–129} }
 @article{tang_knox_nevzorov_2012, title={A spectroscopic assignment technique for membrane proteins reconstituted in magnetically aligned bicelles}, volume={54}, ISSN={["0925-2738"]}, DOI={10.1007/s10858-012-9673-y}, abstractNote={Oriented-sample NMR (OS-NMR) has emerged as a powerful tool for the structure determination of membrane proteins in their physiological environments. However, the traditional spectroscopic assignment method in OS NMR that uses the "shotgun" approach, though effective, is quite labor- and time-consuming as it is based on the preparation of multiple selectively labeled samples. Here we demonstrate that, by using a combination of the spin exchange under mismatched Hartmann-Hahn conditions and a recent sensitivity-enhancement REP-CP sequence, spectroscopic assignment of solid-state NMR spectra of Pf1 coat protein reconstituted in magnetically aligned bicelles can be significantly improved. This method yields a two-dimensional spin-exchanged version of the SAMPI4 spectrum correlating the (15)N chemical shift and (15)N-(1)H dipolar couplings, as well as spin-correlations between the (i, i ± 1) amide sites. Combining the spin-exchanged SAMPI4 spectrum with the original SAMPI4 experiment makes it possible to establish sequential assignments, and this technique is generally applicable to other uniaxially aligned membrane proteins. Inclusion of an (15)N-(15)N correlation spectrum into the assignment process helps establish correlations between the peaks in crowded or ambiguous spectral regions of the spin-exchanged SAMPI4 experiment. Notably, unlike the traditional method, only a uniformly labeled protein sample is required for spectroscopic assignment with perhaps only a few selectively labeled "seed" spectra. Simulations for the magnetization transfer between the dilute spins under mismatched Hartmann Hahn conditions for various B (1) fields have also been performed. The results adequately describe the optimal conditions for establishing the cross peaks, thus eliminating the need for lengthy experimental optimizations.}, number={3}, journal={JOURNAL OF BIOMOLECULAR NMR}, author={Tang, Wenxing and Knox, Robert W. and Nevzorov, Alexander A.}, year={2012}, month={Nov}, pages={307–316} }
 @article{tang_yin_nevzorov_2012, title={NMR Spectroscopic Assignment and Structure Determination of Membrane Proteins Reconstituted in Magnetically Aligned Bicelles}, volume={102}, ISSN={0006-3495}, url={http://dx.doi.org/10.1016/j.bpj.2011.11.2132}, DOI={10.1016/j.bpj.2011.11.2132}, abstractNote={Magnetically aligned bicelles represent a nearly ideal mimetic for the structural studies of membrane proteins in their native lipid environment. Structures of several proteins reconstituted in bicelles have been obtained by solid-state NMR, including MerF, VPU, and Pf1. However, the assignment of NMR spectra of membrane proteins is usually performed by a laborious preparation of multiple selectively labeled samples, and a helical wheel-like arrangement of peaks is assumed. Here we have used a novel spectroscopic technique [1,2] to obtain spin correlations between the resonances and assigned the spectrum of uniformly 15N labeled Pf1 protein reconstituted in aligned bicelles. The peaks generally follow the (i, i+1) connectivity pattern, and yield the assignment identical to the previously established, albeit without using multiple labeled samples [3]. The method is generally applicable to membrane proteins of arbitrary topology. Long-range correlations (up to 6.7 Å) can be observed that have a potential for establishing spectroscopic contacts between the neighboring transmembrane helices. Furthermore, we have developed an algorithm [4] that calculates three-dimensional structures of membrane proteins solely based on the dipolar couplings between the covalently bound spins. Structures of protein G and a helical hairpin of bacteriorhodopsin have been calculated from simulated solid-state NMR spectra as illustrative examples. Effect of experimental uncertainty has been assessed. Application of the algorithm to fully assigned three-dimensional NMR data of doubly (15N, 13C) labeled membrane proteins may allow one to obtain the entire backbone structure from a single spectrum. [1] A.A. Nevzorov, J. Am. Chem. Soc. 130 (2008) 11282-11283. [2] R.W. Knox, J. Lu, S.J. Opella, A.A. Nevzorov, J. Am. Chem. Soc. 132 (2010) 8255-8257. [3] S.J. Opella, A.C. Zeri, S.H. Park, Annu. Rev. Phys. Chem. 59 (2008) 635-57. [4] Y.Y. Yin, A.A. Nevzorov, J. Magn. Reson. 212 (2011) 64-73.}, number={3}, journal={Biophysical Journal}, publisher={Elsevier BV}, author={Tang, Wenxing and Yin, Yuanyuan and Nevzorov, Alexander}, year={2012}, month={Jan}, pages={390a} }
 @article{nevzorov_2011, title={Ergodicity and efficiency of cross-polarization in NMR of static solids}, volume={209}, ISSN={["1090-7807"]}, DOI={10.1016/j.jmr.2011.01.006}, abstractNote={Cross-polarization transfer is employed in virtually every solid-state NMR experiment to enhance magnetization of low-gamma spins. Theory and experiment is used to assess the magnitude of the final quasistationary magnetization amplitude. The many-body density matrix equation is solved for relatively large (up to N = 14) spin systems without the spin-temperature assumption for the final spin states. Simulations show that about 13% of the thermodynamic limit is still retained within the proton bath. To test this theoretical prediction, a combination of a reverse cross-polarization experiment and multiple contacts is employed to show that the thermodynamic limit of magnetization cannot be transferred from high- to low-gamma nuclei in a single contact. Multiple contacts, however, fully transfer the maximum magnetization. A simple diffusion on a cone model shows that slow dynamics can affect the build up profile for the transferred magnetization.}, number={2}, journal={JOURNAL OF MAGNETIC RESONANCE}, author={Nevzorov, Alexander A.}, year={2011}, month={Apr}, pages={161–166} }
 @article{peng_desousa_su_novak_nevzorov_garland_melander_2011, title={Inhibition of Acinetobacter baumannii biofilm formation on a methacrylate polymer containing a 2-aminoimidazole subunit}, volume={47}, ISSN={1359-7345 1364-548X}, url={http://dx.doi.org/10.1039/c1cc10691k}, DOI={10.1039/c1cc10691k}, abstractNote={A polymeric composite containing a 2-aminoimidazole derivative was synthesized. It was found that this polymer was resistant to biofilm colonization by Acinetobacter baumannii, no leaching of the 2-aminoimidazole derivative was observed after 2 weeks of treatment with deionized water, and the resulting polymer was not hemolytic.}, number={17}, journal={Chemical Communications}, publisher={Royal Society of Chemistry (RSC)}, author={Peng, Lingling and DeSousa, Joseph and Su, Zhaoming and Novak, Bruce M. and Nevzorov, Alexander A. and Garland, Eva R. and Melander, Christian}, year={2011}, pages={4896} }
 @article{nevzorov_2011, title={Orientational and Motional Narrowing of Solid-State NMR Lineshapes of Uniaxially Aligned Membrane Proteins}, volume={115}, ISSN={["1520-6106"]}, DOI={10.1021/jp2092847}, abstractNote={A unified theory for the NMR line shapes of aligned membrane proteins arising from uniaxial disorder (mosaic spread) and global rotational diffusion about the director axis is presented. A superoperator formalism allows one to take into account the effects of continuous radiofrequency irradiation and frequency offsets in the presence of dynamics. A general method based on the Stochastic Liouville Equation makes it possible to bridge the static and dynamic limits in a single model. Simulations of solid-state NMR spectra are performed for a uniform α helix by considering orientational disorder and diffusion of the helix as a whole relative to the alignment axis. The motional narrowing of the resonance lines is highly inhomogeneous and can be used as an additional angular restraint in structure calculations. Experimental solid-state NMR spectra of Pf1 coat protein support the conclusions of the theory for two limiting cases. The static disorder dominates the (15)N NMR spectra of Pf1 aligned on a phage, while fast uniaxial diffusion provides a line narrowing mechanism for the Pf1 protein reconstituted in magnetically aligned bicelles.}, number={51}, journal={JOURNAL OF PHYSICAL CHEMISTRY B}, author={Nevzorov, Alexander A.}, year={2011}, month={Dec}, pages={15406–15414} }
 @article{tang_nevzorov_2011, title={Repetitive cross-polarization contacts via equilibration-re-equilibration of the proton bath: Sensitivity enhancement for NMR of membrane proteins reconstituted in magnetically aligned bicelles}, volume={212}, ISSN={["1090-7807"]}, DOI={10.1016/j.jmr.2011.06.028}, abstractNote={Thermodynamic limit of magnetization corresponding to the intact proton bath usually cannot be transferred in a single cross-polarization contact. This is mainly due to the finite ratio between the number densities of the high- and low-gamma nuclei, quantum-mechanical bounds on spin dynamics, and Hartmann-Hahn mismatches due to rf field inhomogeneity. Moreover, for fully hydrated membrane proteins refolded in magnetically oriented bicelles, short spin-lock relaxation times (T1ρ) and rf heating can further decrease cross polarization efficiency. Here we show that multiple equilibrations-re-equilibrations of the high- and low-spin reservoirs during the preparation period yield an over twofold gain in the magnetization transfer as compared to a single-contact cross polarization (CP), and up to 45% enhancement as compared to the mismatch-optimized CP-MOIST scheme for bicelle-reconstituted membrane proteins. This enhancement is achieved by employing the differences between the spin-lattice relaxation times for the high- and low-gamma spins. The new technique is applicable to systems with short T1ρ's, and speeds up acquisition of the multidimensional solid-state NMR spectra of oriented membrane proteins for their subsequent structural and dynamic studies.}, number={1}, journal={JOURNAL OF MAGNETIC RESONANCE}, author={Tang, Wenxing and Nevzorov, Alexander A.}, year={2011}, month={Sep}, pages={245–248} }
 @article{yin_nevzorov_2011, title={Structure determination in "shiftless" solid state NMR of oriented protein samples}, volume={212}, ISSN={["1096-0856"]}, DOI={10.1016/j.jmr.2011.06.008}, abstractNote={An efficient formalism for calculating protein structures from oriented-sample NMR data in the torsion-angle space is presented. Angular anisotropies of the NMR observables are treated by utilizing an irreducible spherical basis of rotations. An intermediate rotational transformation is introduced that greatly speeds up structural fitting by rendering the dependence on the torsion angles Φ and Ψ in a purely diagonal form. Back-calculation of the simulated solid-state NMR spectra of protein G involving 15N chemical shift anisotropy (CSA), and 1H-15N and 1Hα-13Cα dipolar couplings was performed by taking into account non-planarity of the peptide linkages and experimental uncertainty. Even a relatively small (to within 1 ppm) random variation in the CSA values arising from uncertainties in the tensor parameters yields the RMSD's of the back-calculated structures of more than 10 Å. Therefore, the 15N CSA has been substituted with heteronuclear dipolar couplings which are derived from the highly conserved bond lengths and bond angles associated with the amino-acid covalent geometry. Using the additional 13Cα-15N and 13C'-15N dipolar couplings makes it possible to calculate protein structures entirely from "shiftless" solid-state NMR data. With the simulated "experimental" uncertainty of 15 Hz for protein G and 120 Hz for a helical hairpin derived from bacteriorhodopsin, back-calculation of the synthetic dipolar NMR spectra yielded a converged set of solutions. The use of distance restraints dramatically improves structural convergence even if larger experimental uncertainties are assumed.}, number={1}, journal={JOURNAL OF MAGNETIC RESONANCE}, author={Yin, Yuanyuan and Nevzorov, Alexander A.}, year={2011}, month={Sep}, pages={64–73} }
 @article{knox_lu_opella_nevzorov_2010, title={A Resonance Assignment Method for Oriented-Sample Solid-State NMR of Proteins}, volume={132}, ISSN={0002-7863 1520-5126}, url={http://dx.doi.org/10.1021/ja102932n}, DOI={10.1021/ja102932n}, abstractNote={A general sequential assignment strategy for uniformly (15)N-labeled uniaxially aligned membrane proteins is proposed. Mismatched Hartmann-Hahn magnetization transfer is employed to establish proton-mediated correlations among the neighboring (15)N backbone spins. Magnetically aligned Pf1 phage coat protein was used to illustrate the method. Exchanged and nonexchanged separated local field spectra were acquired and overlaid to distinguish the cross-peaks from the main peaks. Most of the original assignments from the literature were confirmed without selectively labeled samples. This method is applicable to proteins with arbitrary topology and will find use in assigning solid-state NMR spectra of oriented membrane proteins for their subsequent structure determination.}, number={24}, journal={Journal of the American Chemical Society}, publisher={American Chemical Society (ACS)}, author={Knox, Robert W. and Lu, George J. and Opella, Stanley J. and Nevzorov, Alexander A.}, year={2010}, month={Jun}, pages={8255–8257} }
 @article{nevzorov_2009, title={High-resolution local field spectroscopy with internuclear correlations}, volume={201}, ISSN={["1096-0856"]}, DOI={10.1016/j.jmr.2009.08.006}, abstractNote={Establishing correlations among distant (>3A) spins remains an outstanding problem for both spectral assignment and elucidation of interhelical contacts in solid-state NMR of oriented membrane proteins. Here we present a pulse sequence which incorporates the previously established mismatched Hartmann-Hahn mixing of dilute spins via the proton bath together with high-resolution local field spectroscopy. In addition to providing structural information, the use of dipolar couplings in the indirect dimension helps eliminate the spectral crowdedness compared to the standard homonuclear correlation techniques. The proposed pulse sequence may find its use in assigning protein spectra in uniaxially oriented membrane environments.}, number={1}, journal={JOURNAL OF MAGNETIC RESONANCE}, author={Nevzorov, Alexander A.}, year={2009}, month={Nov}, pages={111–114} }
 @article{nevzorov_2008, title={Mismatched Hartmann-Hahn conditions cause proton-mediated intermolecular magnetization transfer between dilute low-spin nuclei in NMR of static solids}, volume={130}, ISSN={["0002-7863"]}, DOI={10.1021/ja804326b}, abstractNote={Mismatched Hartmann-Hahn conditions between the protons and dilute spins (such as 15N) are found to cause intermolecular magnetization transfer between the low-gamma nuclei over long distances. This transfer is purely proton mediated and occurs even in the absence of direct 15N-15N couplings. This has been demonstrated experimentally using a static single crystal of n-acetyl Leucine with intermolecular distances between the 15N nuclei exceeding 6.5 A. A quantum-mechanical explanation of this phenomenon is given based on the average-Hamiltonian theory which was confirmed by detailed numerical many-spin simulations. The theory and experiment presented in the present paper may help in the development of solid-state NMR methods for studying interhelical contacts in membrane proteins, as well as for their spectral assignment.}, number={34}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Nevzorov, Alexander A.}, year={2008}, month={Aug}, pages={11282-+} }
 @article{nevzorov_opella_2007, title={Selective averaging for high-resolution solid-state NMR spectroscopy of aligned samples}, volume={185}, ISSN={1090-7807}, url={http://dx.doi.org/10.1016/j.jmr.2006.09.006}, DOI={10.1016/j.jmr.2006.09.006}, abstractNote={Solid-state NMR experiments benefit from being performed at high fields, and this is essential in order to obtain spectra with the resolution and sensitivity required for applications to protein structure determination in aligned samples. Since the amount of rf power that can be applied is limited, especially for aqueous protein samples, the most important pulse sequences suffer from bandwidth limitations resulting from the same spread in chemical shift frequencies that aids resolution. SAMPI4 is a pulse sequence that addresses these limitations. It yields separated local field spectra with narrower and more uniform linewidths over the entire spectrum than the currently used PISEMA and SAMMY experiments. In addition, it is much easier to set up on commercial spectrometers and can be incorporated as a building block into other multidimensional pulse sequences. This is illustrated with a two-dimensional HETCOR experiment, where it is crucial to transfer polarization from the amide protons to their directly bonded nitrogens over a wide range of chemical shift frequencies. A quantum-mechanical treatment of the spin Hamiltonians under high-power rf pulses is presented which gives the scaling factor for SAMPI4 as well as the durations of the rf pulses to achieve optimal decoupling.}, number={1}, journal={Journal of Magnetic Resonance}, publisher={Elsevier BV}, author={Nevzorov, Alexander A. and Opella, Stanley J.}, year={2007}, month={Mar}, pages={59–70} }
 @misc{brown_heyn_job_kim_moltke_nakanishi_nevzorov_struts_salgado_wallat_2007, title={Solid-State H-2 NMR spectroscopy of retinal proteins in aligned membranes}, volume={1768}, ISSN={["0005-2736"]}, DOI={10.1016/j.bbamem.2007.10.014}, abstractNote={Solid-state 2H NMR spectroscopy gives a powerful avenue to investigating the structures of ligands and cofactors bound to integral membrane proteins. For bacteriorhodopsin (bR) and rhodopsin, retinal was site-specifically labeled by deuteration of the methyl groups followed by regeneration of the apoprotein. 2H NMR studies of aligned membrane samples were conducted under conditions where rotational and translational diffusion of the protein were absent on the NMR time scale. The theoretical lineshape treatment involved a static axial distribution of rotating C-C2H3 groups about the local membrane frame, together with the static axial distribution of the local normal relative to the average normal. Simulation of solid-state 2H NMR lineshapes gave both the methyl group orientations and the alignment disorder (mosaic spread) of the membrane stack. The methyl bond orientations provided the angular restraints for structural analysis. In the case of bR the retinal chromophore is nearly planar in the dark- and all-trans light-adapted states, as well upon isomerization to 13-cis in the M state. The C13-methyl group at the "business end" of the chromophore changes its orientation to the membrane upon photon absorption, moving towards W182 and thus driving the proton pump in energy conservation. Moreover, rhodopsin was studied as a prototype for G protein-coupled receptors (GPCRs) implicated in many biological responses in humans. In contrast to bR, the retinal chromophore of rhodopsin has an 11-cis conformation and is highly twisted in the dark state. Three sites of interaction affect the torsional deformation of retinal, viz. the protonated Schiff base with its carboxylate counterion; the C9-methyl group of the polyene; and the beta-ionone ring within its hydrophobic pocket. For rhodopsin, the strain energy and dynamics of retinal as established by 2H NMR are implicated in substituent control of activation. Retinal is locked in a conformation that is twisted in the direction of the photoisomerization, which explains the dark stability of rhodopsin and allows for ultra-fast isomerization upon absorption of a photon. Torsional strain is relaxed in the meta I state that precedes subsequent receptor activation. Comparison of the two retinal proteins using solid-state 2H NMR is thus illuminating in terms of their different biological functions.}, number={12}, journal={BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES}, author={Brown, Michael F. and Heyn, Maarten P. and Job, ConstAntin and Kim, Suhkmann and Moltke, Stephan and Nakanishi, Koji and Nevzorov, Alexander A. and Struts, Andrey V. and Salgado, Gilmar F. J. and Wallat, Ingrid}, year={2007}, month={Dec}, pages={2979–3000} }
 @article{nevzorov_park_opella_2007, title={Three-dimensional experiment for solid-state NMR of aligned protein samples in high field magnets}, volume={37}, ISSN={0925-2738 1573-5001}, url={http://dx.doi.org/10.1007/s10858-006-9121-y}, DOI={10.1007/s10858-006-9121-y}, abstractNote={A pulse sequence that yields three-dimensional (1)H chemical shift/(1)H-(15)N heteronuclear dipolar coupling/(15)N chemical shift solid-state NMR spectra is demonstrated on a uniformly (15)N labeled membrane protein in magnetically aligned phospholipid bilayers. Based on SAMPI4, the pulse sequence yields high resolution in all three dimensions at a (1)H resonance frequency of 900 MHz with the relatively low rf field strength (33 kHz) available for a lossy aqueous sample with a commercial spectrometer and probe. The (1)H chemical shift frequency dimension is shown to select among amide resonances, which will be useful in studies of larger polytopic membrane proteins where the resonances overlap in two-dimensional spectra. Moreover, the (1)H chemical shift, which can be measured from these spectra, provides an additional orientationally dependent frequency as input for structure calculations.}, number={2}, journal={Journal of Biomolecular NMR}, publisher={Springer Science and Business Media LLC}, author={Nevzorov, Alexander A. and Park, Sang Ho and Opella, Stanley J.}, year={2007}, month={Jan}, pages={113–116} }