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Recent structural investigation of amyloid filaments extracted from human patients demonstrated that the ex vivo filaments associated with different disease phenotypes adopt diverse molecular conformations, which are different from those of in vitro amyloid filaments. A very recent cryo-EM structural study also revealed that ex vivo α-synuclein filaments extracted from multiple system atrophy patients adopt distinct molecular structures from those of in vitro α-synuclein filaments, suggesting the presence of co-factors for α-synuclein aggregation in vivo. Here, we report structural characterizations of α-synuclein filaments formed in the presence of a potential co-factor, tau, using cryo-EM and solid-state NMR. Our cryo-EM structure of the tau-promoted α-synuclein filaments reveals some similarities to one of the previously reported polymorphs of in vitro α-synuclein filaments in the core region, while illustrating distinct conformations in the N- and C-terminal regions. The structural study highlights the conformational plasticity of α-synuclein filaments and the importance of the co-factors, requiring additional structural investigation of not only more ex vivo α-synuclein filaments, but also in vitro α-synuclein filaments formed in the presence of diverse co-factors. The comparative structural analyses will help better understand molecular basis of diverse structures of α-synuclein filaments and possible relevance of each structure to the disease phenotype.

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The electronic g factor carries highly useful information about the electronic structure of a paramagnetic species, such as spin-orbit coupling and dia- or paramagnetic (de-)shielding due to local fields of surrounding electron pairs. However, in many cases, a near “spin-only” case is observed, in particular for light elements, necessitating accurate and precise measurement of the g factors. Such measurement is typically impeded by a “chicken and egg situation”: internal or external reference standards are used for relative comparison of electron paramagnetic resonance (EPR) Larmor frequencies. However, the g factor of the standard itself usually is subject to a significant uncertainty which directly limits the precision and/or accuracy of the sought after sample g factor. Here, we apply an EPR reference-free approach for determining the g factor of atomic nitrogen trapped within the endohedral fullerene C60:N@C60 in its polycrystalline state by measuring the 1H NMR resonance frequency of dispersing toluene at room temperature. We found a value of g = 2.00204 ( 4 ) with a finally reached relative precision of ∼20 ppm. This accurate measurement allows us to directly compare the electronic properties of N@C60 to those found in atomic nitrogen in the gas phase or trapped in other solid matrices at liquid helium temperature. We conclude that spin-orbit coupling in N@C60 at room temperature is very similar in magnitude and of same sign as found in other inert solid matrices and that interactions between the quartet spin system and the C60 molecular orbitals are thus negligible.

Chloride ions play important roles in many chemical and biological processes. This paper investigates the possibility of localizing 35Cl nuclei using solid-state NMR. It demonstrates that distances shorter than 3.8A, between 13C atoms and 35Cl atoms in 10% uniformly labeled 13C L-tyrosine·HCl and natural abundance Glycine·HCl can be measured using rotational-echo (adiabatic passage) double-resonance (RE(AP)DOR). Furthermore the effect of quadrupolar interaction on the REDOR/REAPDOR experiment is quantified. The dephasing curve is plotted in a three dimensional chart as a function of the dephasing time and of the strength of quadrupolar interaction felt by each orientation. During spinning each orientation feels a quadrupolar interaction that varies in time, and therefore at each moment in time we reorder the crystallite orientations as a function of their contribution to the dephasing curve. In this way the effect of quadrupolar interaction on the dipolar dephasing curve can be fitted with a polynomial function. The numerical investigation performed allows us to generate REDOR/REAPDOR curves which are then used to simulate the experimental data.

The paper describes a family of novel recoupling pulse sequences, called three pulse recoupling. These pulse sequences can be employed for both homonuclear and heteronuclear recoupling experiments and are robust to dispersion in chemical shifts and rf-inhomogeneity. These recoupling pulse sequences can be used in design of two-dimensional solid state NMR experiments that use powdered dephased antiphase coherence (γ preparation) to encode chemical shifts in the indirect dimension. Both components of this chemical shift encoded gamma-prepared states can be refocused into inphase coherence by a recoupling element. This helps to achieve sensitivity enhancement in 2D NMR experiments by quadrature detection.

Biological processes occur in complex environments containing a myriad of potential interactors. Unfortunately, limitations on the sensitivity of biophysical techniques normally restrict structural investigations to purified systems, at concentrations that are orders of magnitude above endogenous levels. Dynamic nuclear polarization (DNP) can dramatically enhance the sensitivity of nuclear magnetic resonance (NMR) spectroscopy and enable structural studies in biologically complex environments. Here, we applied DNP NMR to investigate the structure of a protein containing both an environmentally sensitive folding pathway and an intrinsically disordered region, the yeast prion protein Sup35. We added an exogenously prepared isotopically labeled protein to deuterated lysates, rendering the biological environment “invisible” and enabling highly efficient polarization transfer for DNP. In this environment, structural changes occurred in a region known to influence biological activity but intrinsically disordered in purified samples. Thus, DNP makes structural studies of proteins at endogenous levels in biological contexts possible, and such contexts can influence protein structure., United States. National Institutes of Health (GM-025874), United States. National Institutes of Health (EB-003151), United States. National Institutes of Health (EB-002804), United States. National Institutes of Health (EB-002026)

Modular and compact adsorption heat pumps (AHPs) promise an energy-efficient alternative to conventional vapor compression based heating, ventilation and air conditioning systems. A key element in the advancement of AHPs is the development of adsorbents with high uptake capacity, fast intracrystalline diffusivity and durable hydrothermal stability. Herein, the ion exchange of NaY zeolites with ingoing Mg[superscript 2+] ions is systematically studied to maximize the ion exchange degree (IED) for improved sorption performance. It is found that beyond an ion exchange threshold of 64.1%, deeper ion exchange does not benefit water uptake capacity or characteristic adsorption energy, but does enhance the vapor diffusivity. In addition to using water as an adsorbate, the uptake properties of Mg, Na-Y zeolites were investigated using 20 wt.% MeOH aqueous solution as a novel anti-freeze adsorbate, revealing that the MeOH additive has an insignificant influence on the overall sorption performance. We also demonstrated that the lab-scale synthetic scalability is robust, and that the tailored zeolites scarcely suffer from hydrothermal stability even after successive 108-fold adsorption/desorption cycles. The samples were analyzed using N[superscript 2] sorption, [superscript 27]Al/[superscript 29]Si MAS NMR spectroscopy, ICP-AES, dynamic vapor sorption, SEM, Fick’s 2nd law and D–R equation regressions. Among these, close examination of sorption isotherms for H[subscript 2]O and N[subscript 2] adsorbates allows us to decouple and extract some insightful information underlying the complex water uptake phenomena. This work shows the promising performance of our modified zeolites that can be integrated into various AHP designs for buildings, electronics, and transportation applications., United States. Advanced Research Projects Agency-Energy (0471-1627), National Institute for Biomedical Imaging and Bioengineering (U.S.) (Awards EB-001960 and EB-002026), Natural Sciences and Engineering Research Council of Canada (Postgraduate Fellowship)

National Institutes of Health (U.S.) (EB-002804), National Institutes of Health (U.S.) (EB-002026)

Specific, high affinity protein-protein interactions lie at the heart of many essential biological processes, including the recognition of an apparently limitless range of foreign proteins by natural antibodies, which has been exploited to develop therapeutic antibodies. To mediate biological processes, high affinity protein complexes need to form on appropriate, relatively rapid timescales, which presents a challenge for the productive engagement of complexes with large and complex contact surfaces (∼600–1800 Å2). We have obtained comprehensive backbone NMR assignments for two distinct, high affinity antibody fragments (single chain variable and antigen-binding (Fab) fragments), which recognize the structurally diverse cytokines interleukin-1β (IL-1β, β-sheet) and interleukin-6 (IL-6, α-helical). NMR studies have revealed that the hearts of the antigen binding sites in both free anti-IL-1β Fab and anti-IL-6 single chain variable exist in multiple conformations, which interconvert on a timescale comparable with the rates of antibody-antigen complex formation. In addition, we have identified a conserved antigen binding-induced change in the orientation of the two variable domains. The observed conformational heterogeneity and slow dynamics at protein antigen binding sites appears to be a conserved feature of many high affinity protein-protein interfaces structurally characterized by NMR, suggesting an essential role in protein complex formation. We propose that this behavior may reflect a soft capture, protein-protein docking mechanism, facilitating formation of high affinity protein complexes on a timescale consistent with biological processes.

We present a new, efficient strategy for designing fully balanced transmission line RF circuits for solid state NMR probes based on back propagation of common impedance nodes (BPCIN). In this approach, the impedance node phenomenon is the sole means of achieving mutual RF isolation and balance in all RF channels. BPCIN is illustrated using a custom double resonance 3.2 mm MAS probe operating at 500 MHz ([superscript 1]H) and 125 MHz ([superscript 13]C). When fully optimized, the probe is capable of producing high homogeneity (810°/90° ratios of 86% and 89% for 1H and 13C, respectively) and high efficiency (γB[superscript 1] = 100 kHz for [superscript 1]H and [superscript 13]C at 70 W and 180 W of RF input, respectively; up to 360 kHz for [superscript 1]H). The probe’s performance is illustrated by 2D MAS correlation spectra of microcrystals of the tripeptide N-f-MLF-OH and hydrated amyloid fibrils of the protein PI3-SH3., National Institutes of Health (U.S.) (NIH Grant EB003151), National Institutes of Health (U.S.) (NIH grant EB002026), National Institutes of Health (U.S.) (NIH grant EB001960), National Institutes of Health (U.S.) (NIH grant EB001035)

We describe the design and implementation of the instrumentation required to perform DNP-NMR at higher field strengths than previously demonstrated, and report the first magic-angle spinning (MAS) DNP-NMR experiments performed at ¹H/e⁻ frequencies of 700 MHz/460 GHz. The extension of DNP-NMR to 16.4 T has required the development of probe technology, cryogenics, gyrotrons, and microwave transmission lines. The probe contains a 460 GHz microwave channel, with corrugated waveguide, tapers, and miter-bends that couple microwave power to the sample. Experimental efficiency is increased by a cryogenic exchange system for 3.2 mm rotors within the 89 mm bore. Sample temperatures ⩽85 K, resulting in improved DNP enhancements, are achieved by a novel heat exchanger design, stainless steel and brass vacuum jacketed transfer lines, and a bronze probe dewar. In addition, the heat exchanger is preceded with a nitrogen drying and generation system in series with a pre-cooling refrigerator. This reduces liquid nitrogen usage from >700 l per day to 7 days) cryogenic spinning without detrimental frost or ice formation. Initial enhancements, ε = −40, and a strong microwave power dependence suggests the possibility for considerable improvement. Finally, two-dimensional spectra of a model system demonstrate that the higher field provides excellent resolution, even in a glassy, cryoprotecting matrix., National Institutes of Health (U.S.) (EB002804), National Institutes of Health (U.S.) (EB003151), National Institutes of Health (U.S.) (EB002026), National Institutes of Health (U.S.) (EB001960), National Institutes of Health (U.S.) (EB001035), National Institutes of Health (U.S.) (EB001965), National Institutes of Health (U.S.) (EB004866)

We described a versatile spectrometer designed for the study of dynamic nuclear polarization (DNP) at low temperatures and high fields. The instrument functions both as an NMR spectrometer operating at 212 MHz ([superscript 1]H frequency) with DNP capabilities, and as a pulsed-EPR operating at 140 GHz. A coiled TE[subscript 011] resonator acts as both an NMR coil and microwave resonator, and a double balanced ([superscript 1]H, [superscript 13]C) radio frequency circuit greatly stabilizes the NMR performance. A new 140 GHz microwave bridge has also been developed, which utilizes a four-phase network and ELDOR channel at 8.75 GHz, that is then multiplied and mixed to obtain 140 GHz microwave pulses with an output power of 120 mW. Nutation frequencies obtained are as follows: 6 MHz on S = 1/2 electron spins, 100 kHz on [superscript 1]H, and 50 kHz on [superscript 13]C. We demonstrate basic EPR, ELDOR, ENDOR, and DNP experiments here. Our solid effect DNP results demonstrate an enhancement of 144 and sensitivity gain of 310 using OX063 trityl at 80 K and an enhancement of 157 and maximum sensitivity gain of 234 using Gd-DOTA at 20 K, which is significantly better performance than previously reported at high fields (⩾3 T)., National Institutes of Health (U.S.) (EB002804), National Institutes of Health (U.S.) (EB002026), National Institutes of Health (U.S.) (EB001965), National Institutes of Health (U.S.) (EB004866), Deutsche Forschungsgemeinschaft (Postdoctoral Fellowship)

We describe the design and implementation of a novel tunable 250 GHz gyrotron oscillator with >10 W output power over most of a 3 GHz band and >35 W peak power. The tuning bandwidth and power are sufficient to generate a >1 MHz nutation frequency across the entire nitroxide EPR lineshape for cross effect DNP, as well as to excite solid effect transitions utilizing other radicals, without the need for sweeping the NMR magnetic field. Substantially improved tunability is achieved by implementing a long (23 mm) interaction cavity that can excite higher order axial modes by changing either the magnetic field of the gyrotron or the cathode potential. This interaction cavity excites the rotating TE[subscript 5,2,q] mode, and an internal mode converter outputs a high-quality microwave beam with >94% Gaussian content. The gyrotron was integrated into a DNP spectrometer, resulting in a measured DNP enhancement of 54 on the membrane protein bacteriorhodopsin., National Institutes of Health (U.S.) (Grant EB002804), National Institutes of Health (U.S.) (Grant EB003151), National Institutes of Health (U.S.) (Grant EB002026), National Institutes of Health (U.S.) (Grant EB001960), National Institutes of Health (U.S.) (Grant EB001035), National Institutes of Health (U.S.) (Grant EB001965), National Institutes of Health (U.S.) (Grant EB004866), National Science Foundation (U.S.). Graduate Research Fellowship

The paper describes two-dimensional solid state NMR experiments that use powdered dephased anti-phase coherence (γ preparation) to encode chemical shifts in the indirect dimension. Both components of this chemical shift encoded gamma-prepared states can be refocused into inphase coherence by a recoupling element. This helps to achieve sensitivity enhancement in 2D NMR experiments by quadrature detection. The powder dependence of the gamma-prepared states allows for manipulating them by suitable insertion of delays in the recoupling periods. This helps to design experiments that suppress diagonal peaks in 2D spectra, leading to improved resolution. We describe some new phase modulated heteronuclear and homonuclear recoupling pulse sequences that simplify the implementation of the described experiments based on γ prepared states. Recoupling in the heteronuclear spin system is achieved by matching the difference in the amplitude of the sine/cosine modulated phase on the two rf-channels to the spinning frequency while maintaining the same power on the two rf-channels.

Gas vesicles are gas-filled buoyancy organelles with walls that consist almost exclusively of gas vesicle protein A (GvpA). Intact, collapsed gas vesicles from the cyanobacterium Anabaena flos-aquae were studied by solid-state NMR spectroscopy, and most of the GvpA sequence was assigned. Chemical shift analysis indicates a coil-α-β-β-α-coil peptide backbone, consistent with secondary-structure-prediction algorithms, and complementary information about mobility and solvent exposure yields a picture of the overall topology of the vesicle subunit that is consistent with its role in stabilizing an air-water interface.

We present a de novo high-resolution structure of the peptide Alanyl-Prolyl-Glycine using a combination of sensitive solid-state NMR techniques that each yield precise structural constraints. High-quality (13)C-(13)C distance constraints are extracted by fitting rotational resonance width (R(2)W) experiments using Multimode Multipole Floquet Theory and experimental chemical shift anisotropy (CSA) orientations. In this strategy, a structure is first calculated using DANTE-REDOR and torsion angle measurements and the resulting relative CSA orientations are used as an input parameter in the (13)C-(13)C distance calculations. Finally, a refined structure is calculated using all the constraints. We investigate the effect of different structural constraints on structure quality, as determined by comparison to the crystal structure and also self-consistency of the calculated structures. Inclusion of all or subsets of these constraints into CNS calculations resulted in high-quality structures (0.02A backbone RMSD using all 11 constraints).

We describe a field-lock/field-sweep system for the use in superconducting magnets. The system is based on a commercially available field mapping unit and a custom designed broad-band 1 H NMR probe. The NMR signal of a small water sample is used in a feedback loop to set and control the magnetic field to high accuracy. The current instrumental configuration allows field sweeps of ±0.4 T and a resolution of up to 10 −5 T (0.1 G) and the performance of the system is demonstrated in a high-field electron paramagnetic resonance (EPR) application. The system should also be of utility in other experiments requiring precise and reproducible sweeps of the magnetic field such as DNP, ENDOR or PELDOR.

Exact numerical simulations of NMR experiments are often required for the development of new techniques and for the extraction of structural and dynamic information from the spectra. Simulations of solid-state magic angle spinning (MAS) experiments can be particularly demanding both computationally and in terms of the programming required to carry them out, even if special simulation software is used. We recently developed a number of approaches that dramatically improve the efficiency and allow a high degree of automation of these computations. In the present paper, we describe SPINEVOLUTION, a highly optimized computer program that implements the new methodology. The algorithms used in the program will be described separately. Although particularly efficient for the simulation of experiments with complex pulse sequences and multi-spin systems in solids, SPINEVOLUTION is a versatile and easy to use tool for the simulation and optimization of virtually any NMR experiment. The performance of SPINEVOLUTION was compared with that of another recently developed NMR simulation package, SIMPSON. Benchmarked on a series of examples, SPINEVOLUTION was consistently found to be orders of magnitude faster. At the time of publication, the program is available gratis for non-commercial use.

N Solid state CP/MAS NMR spectroscopy has been used to study a dinitrogen-derived terminal nitride of molybdenum, 15 NMo(N( t Bu)Ar)3 (Ar = 3,5-(CH3)2C6H3). A number of Lewis acid adducts, including X3E-NMo(N( t Bu)Ar)3 (X =F , E =B ; X = Cl, E = B, Al, Ga, In; X = Br, E = Al; X = I, E = Al) and Cl2E-NMo(N( t Bu)Ar)3 (E = Ge, Sn), were prepared by the combi- nation of 15 NMo(N( t Bu)Ar)3 with 1 equiv. of Lewis acid. A series of cationic imido complexes, (RNMo(N( t Bu)Ar)3)X was prepared by the reaction of electrophiles, RX (R = CH3, X = I; R = PhC(O) or Me3Si, X = OTf (OTf = SO3CF3)), with NMo(N( t Bu)Ar)3. Deprotonation of (CH3NMo(N( t Bu)Ar)3)I by LiN(SiMe3)2 afforded the ketimide complex H2CNMo(N( t Bu)Ar)3, which has been shown to undergo a reaction with neat CH3I to form (CH3CH2NMo(N( t Bu)Ar)3)I. 15 N solid state CP/MAS NMR spectroscopy was employed in the characterization of each complex. Complementary density functional theory (DFT) studies of 15 NMo(N( t- Bu)Ar)3 and derivatives enabled a detailed examination of the experimental solid state NMR parameters in terms of electronic struc- ture at the labeled N-atom. Computational analysis demonstrated that significant paramagnetic contributions to the perpendicular components of the chemical shielding tensor (d11 and d22) were responsible for the huge span of the tensor measured for 15 NMo(N( t- Bu)Ar)3 (X = 1187 ppm). Perturbation of the electronic structure in 15 NMo(N( t Bu)Ar)3 upon coordination of a Lewis acid or for- mation of a cationic imido complex was attributed to stabilization of a r-symmetric orbital. An upfield shift in the perpendicular components of the chemical shift tensor results from the reduced paramagnetic contribution to these tensor components upon increasing the energy gap between the magnetically coupled occupied and virtual orbitals (eoccevir). � 2004 Elsevier Ltd. All rights reserved.

Several single-scan experiments for the measurement of the longitudinal relaxation time (T1) are proposed. These experiments result in fast and accurate determinations of the relaxation rate, are relatively robust to pulse imperfections, and preserve information about the chemical shift. The method used in these experiments is to first encode the T1 values as a spatial variation of the magnetization and then to read out this variation either by applying a weak gradient during acquisition or by sequentially observing different slices of the sample. As a result, it is possible to reduce the time necessary to determine the T1 values by one or two orders of magnitude. This time saving comes at the expense of the signal-to-noise level of the resulting spectrum and some chemical shift resolution.

The solid state NMR technique of rotational resonance ( R 2 ) has been used extensively to measure distances approaching 5–6 A between 13 C nuclei in a variety of compounds including amyloidogenic peptides and membrane proteins. The accuracy of the distance information extracted from the time-dependent spin dynamics at R 2 is often limited by the accuracy with which the relevant zero-quantum lineshape parameters are estimated. Here we demonstrate that measurement of spinning frequency dependent magnetization exchange dynamics provides data from which both distance and zero-quantum relaxation parameters can be extracted independently. In addition to providing more accurate distance information, this technique allows examination of the zero-quantum lineshape, which can indicate the presence of correlated relaxation or chemical shift distributions between dipolar-coupled sites. With this approach we have separated the contribution of dipolar couplings and zero quantum relaxation to R 2 exchange curves. Thus, we have significantly improved the accuracy of the measurement of the intramolecular, internuclear distances between a pair of 13 C ’s in two model compounds ( N -acetyl- d , l -valine and glycylglycine·HCl) that lie in the distance range 4.6–4.7 A.

In this Communication, we demonstrate the use of deuteration together with back substitution of exchangeable protons as a means of attenuating the strong 1H-1H couplings that broaden 1H magic angle spinning (MAS) spectra of solids. The approach facilitates 15N-1H correlation experiments as well as experiments for the measurement of 1H-1H distances. The distance measurement relies on the excellent resolution in the 1H MAS spectrum and homonuclear double quantum recoupling techniques. The 1H-1H dipolar recoupling can be analyzed in an analytical fashion by fitting the data to a 2- or 3-spin system. The experiments are performed on a sample of the dipeptide N-Ac-Val-Leu-OH, which was synthesized from uniformly [2H, 15N] labeled materials and back-exchanged in H2O.

A proton dynamic nuclear polarization (DNP) NMR signal enhancement (epsilon) close to thermal equilibrium, epsilon = 0.89, has been obtained at high field (B(0) = 5 T, nu(epr) = 139.5 GHz) using 15 mM trityl radical in a 40:60 water/glycerol frozen solution at 11 K. The electron-nuclear polarization transfer is performed in the nuclear rotating frame with microwave irradiation during a nuclear spin-lock pulse. The growth of the signal enhancement is governed by the rotating frame nuclear spin-lattice relaxation time (T(1rho)), which is four orders of magnitude shorter than the nuclear spin-lattice relaxation time (T(1n)). Due to the rapid polarization transfer in the nuclear rotating frame the experiment can be recycled at a rate of 1/T(1rho) and is not limited by the much slower lab frame nuclear spin-lattice relaxation rate (1/T(1n)). The increased repetition rate allowed in the nuclear rotating frame provides an effective enhancement per unit time(1/2) of epsilon(t) = 197. The nuclear rotating frame-DNP experiment does not require high microwave power; significant signal enhancements were obtained with a low-power (20 mW) Gunn diode microwave source and no microwave resonant structure. The symmetric trityl radical used as the polarization source is water-soluble and has a narrow EPR linewidth of 10 G at 139.5 GHz making it an ideal polarization source for high-field DNP/NMR studies of biological systems.

High-frequency electron paramagnetic resonance (EPR) spectroscopy has been performed on a nitroxide spin-labeled peptide in fluid aqueous solution. The peptide, which follows the single letter sequence, was reacted with the methanethiosulfonate spin label at the cysteine sulfur. The spin sensitivity of high-frequency EPR is excellent with less than 20 pmol of sample required to obtain spectra with good signal-to-noise ratios. Simulation of the temperature-dependent spectral lineshapes reveals the existence of local anisotropic motion about the nitroxide N-O bond with a motional anisotropy tau( perpendicular)/tau( parallel) ( identical with N) approaching 2.6 at 306 K. Comparison with previous work on rigidly labeled peptides suggests that the spin label is reorienting about its side-chain tether. This study demonstrates the feasibility of performing 140-GHz EPR on biological samples in fluid aqueous solution.

A technique for amplifying the apparent magnitudes of 13 C‐ 1 H v r /2 pud d/ 3, where dd is the dipolar coupling strength, the and 15 N‐ 1 H dipolar interactions in magic-angle spinning experi- dipolar spectrum is dominated by an uninformative cenments is described. By inserting rotor-synchronized 1807 pulses in terband. For a one-bond N ‐ H dipolar coupling measured the evolution period of a 2D dipolar-chemical shift experiment, with MREV-8 homonuclear decoupling, dd E 4.5 kHz and heteronuclear dipolar couplings are doubled or quadrupled rela- the centerband dominates for spinning speeds as low as vr / tive to the spinning speed. The increased number of dipolar side- 2pE 1.5 kHz. While low spinning speeds are desirable for bands is desirable for retaining structural information in the indi- extracting structural information, the necessity of resolving rectly detected dipolar dimension while resolving inequivalent sites different chemical moieties through isotropic chemical shifts in the isotropic chemical shift dimension at relatively high spinresults in an opposing constraint. Higher spinning speeds ning speeds. This coupling amplification method is incorporated into an experiment that determines the peptide torsion angle f average the chemical shift anisotropy (dCSA) more comthrough the relative orientation of the C a ‐H a and N‐H N bonds. pletely and thus yield better-resolved spectra. This conflict It is shown both experimentally and theoretically that the angular is exacerbated at higher magnetic field strengths, since shift resolution of the measurement is enhanced significantly by the anisotropies scale linearly with the field strength while the selective doubling of the N‐H N coupling. q 1997 Academic Press dipolar couplings are field-independent. Thus, the desirable

A solid-state NMR experiment is presented for measuring Ψ torsion angles in (15N–13Cα–13CO–15N)-labeled peptides. Creation of 13Cα–13CO double-quantum coherence (DQC) is followed by selective evolution under 15N–13C dipolar interactions. The time-course of 13Cα–13CO DQC dephasing is sensitive to the Ψ torsion angle, particularly for values associated with β-sheet secondary structure (120–180°). We apply the technique to glycylglycine⋅HCl and extract a value for Ψ that matches the diffraction-determined value (Ψ=162.1°C) with a precision of ±5°.

High-resolution solid state 17 O NMR spectra of 17 O-enriched compounds were observed with multiple-quantum magic-angle-spinning (MQMAS) experiments. The resolution of the 17 O MQMAS spectra is approximately 30- to 150-fold higher than that found in conventional 17 O MAS spectra, making it possible to detect crystallographically distinct oxygen sites. It is shown that 17 O MQMAS studies are feasible for systems with 17 O quadrupole coupling constants up to 7 MHz, provided that sufficiently high radio-frequency field strengths (> 120 kHz and spinning speeds (∼ 20 kHz) can be achieved.

We demonstrate the utility of a two-pulse sequence in obtaining high-resolution solid state NMR spectra of half-integer quadrupolar nuclei with magic-angle-spinning (MAS). The experiment, which utilizes multiple/single-quantum correlation, was first described in a different form by Frydman and Harwood [J. Am. Chem. Soc. 117 (1995) 5367] and yields high-resolution isotropic NMR spectra where shifts are determined by the sum of resonance offset (chemical shift) and second-order quadrupolar effects. The two-pulse sequence described here is shown to provide a higher and more uniform excitation of multiple-quantum coherence than the three-pulse sequence used previously.

A high-frequency dynamic nuclear polarization (DNP)/electron paramagnetic resonance spectrometer operating at 211 MHz for1H and 140 GHz forg= 2 paramagnetic centers (5 T static field) is described. The salient feature of the instrument is a cyclotron-resonance maser (gyrotron) which generates high-frequency, high-power microwave radiation. This gyrotron, which under conventional operation produces millisecond pulses at kilowatt powers, has been adapted to operate at ∼100 W for 1 to 20 s pulses and in the continuous wave mode at the 10 W power level. Experiments combining DNP with magic-angle spinning (MAS) nuclear magnetic resonance were performed on samples consisting of 2% by weight of the free radical BDPA doped into polystyrene. Room-temperature DNP enhancement factors of 10 for1H and 40 for13C were obtained in the NMR-MAS spectra. Static DNP NMR has also been performed on samples containing nitroxides dissolved in water:glycerol solvent mixtures. Enhancements of approximately 200 have been obtained for low-temperature (14 K)1H NMR. A pulsed/CW EPR spectrometer operating at 140 GHz has been developed in conjunction with the DNP spectrometer. Microwave sources include Gunn-diode oscillators which provide low-power (20 mW) radiation, and the gyrotron, which has been used to deliver higher power levels in pulsed experiments. Results using this spectrometer are presented for continuous-wave and echo-detected EPR, electron spin-echo-envelope modulation (ESEEM), and Fourier-transform EPR.

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The nuclear dipole-dipole coupling is a strong function of internuclear distance, and can therefore be used as a sensitive probe of local molecular structure. In most cases, however, the simultaneous presence of anisotropic chemical shielding and multiple dipolar interactions prevents isolation of the dipole coupling between individual spin pairs. Under these circumstances, it is customary to improve spectral resolution by employing magic angle spinning (MAS) which preserves the isotropic part of the chemical shift interaction but partially or fully averages the desired dipolar interactions. This article reviews NMR methods designed to selectively restore and measure dipolar couplings between like (homonuclear) spins and unlike (heteronuclear) spins in rotating solids. Applications of these methods - rotational resonance (R 2 ), rotational-echo double resonance (REDOR), trnsferred-echo double resonance (TEDOR), dipolar recovery at the magic angle (DRAMA), simple excitation for the dephasing of rotational-echo amplitudes (SEDRA), and rf-driven dipolar recoupling (RFDR) - to investigate the structure of biomolecules are presented.

Previous solid state 13C-NMR studies of bacteriorhodopsin (bR) have inferred the C = N configuration of the retinal-lysine Schiff base linkage from the [14-13C]retinal chemical shift (1-3). Here we verify the interpretation of the [14-13C]-retinal data using the [epsilon-13C]lysine 216 resonance. The epsilon-Lys-216 chemical shifts in bR555 (48 ppm) and bR568 (53 ppm) are consistent with a C = N isomerization from syn in bR555 to anti in bR568. The M photointermediate was trapped at pH 10.0 and low temperatures by illumination of samples containing either 0.5 M guanidine-HCl or 0.1 M NaCl. In both preparations, the [epsilon-13C]Lys-216 resonance of M is 6 ppm downfield from that of bR568. This shift is attributed to deprotonation of the Schiff base nitrogen and is consistent with the idea that the M intermediate contains a C = N anti chromophore. M is the only intermediate trapped in the presence of 0.5 M guanidine-HCl, whereas a second species, X, is trapped in the presence of 0.1 M NaCl. The [epsilon-13C]Lys-216 resonance of X is coincident with the signal for bR568, indicating that X is either C = N anti and protonated or C = N syn and deprotonated.

A novel two-dimensional magic-angle-spinning NMR experiment for monitoring exchange of Zeeman order between homonuclear dipolar-coupled spins at rotational resonance is described. It is demonstrated that Fourier analysis of the magnetization-transfer trajectories leads to characteric powder lineshapes from which information on the dipolar-coupling and chemical-shielding tensors can be extracted. Under certain circumstances the dipolar interaction (and thereby the internuclear distance) can be estimated directly by visual inspection of the powder patterns. Extension of the mixing sequence to include rapid switching of the spinning speed to an off-rotational resonance condition permits detection of high-resolution signals during the sampling period. Various aspects of the experiment are described theoretically and demonstrated experimentally with spectra of 13 C- 13 C spin pairs in zinc acetate and glycine.

By using solid-state NMR spectroscopy, the integrity of the active center of alpha-chymotrypsin was investigated under a variety of nonaqueous conditions. Specifically, 13C cross-polarization/magic angle spinning NMR was used to analyze the ability of alpha-chymotrypsin to stabilize a transition state intermediate analog after freezing, drying, and addition of organic solvents (both anhydrous and hydrated) to the resultant powder. Lyophilization disrupted 42 +/- 5% of the active centers; it was determined that this occurred during drying, as opposed to freezing. Seven anhydrous solvents caused 0-50% additional disruption, which occurred immediately on addition of the solvent to the enzyme powder. The extent of structural integrity loss correlated with the solvent hydrophobicity, indicating that further dehydration, i.e. stripping of water retained by the enzyme during lyophilization, was the cause. Enzyme samples prepared with lyoprotecting additives, sucrose and ammonium sulfate, exhibited varying degrees of stabilization against the drying step of lyophilization. Moreover, when hydrophilic anhydrous solvents, which had the highest propensity to strip bound water, were added to the resultant enzyme powders, no additional damage occurred.

A two-dimensional homonuclear correlation experiment for magic angle spinning NMR spectroscopy of polycrystalline solids is described. The approach involves application of a multiple pulse mixing period that scales chemical shifts to zero, thereby removing the barrier to spin exchange among interacting nuclei. Transfer of spin coherence is achieved by a transverse mixing sequence in which eight π pulses, πx-πx-πy-πy-π−y-π−y-π−x-π−x, are applied per rotor period. Spectra of triply-labeled 1,2,3-13C3-D,L-alanine are presented, which demonstrate correlations among all three carbon spins.

A new dynamic nuclear polarization (DNP) technique utilizing high frequency, pulsed microwaves is described and demonstrated with observation of an Overhauser enhanced, 13 C magic angle spinning nuclear magnetic resonance spectrum of a powdered sample of the organic conductor, fluoranthenyl hexafluorophosphate ((FA) 2 PF 6 ). The spectrum was obtained at room temperature in a 5 T magnetic field, where the conduction electron spin resonance frequency is 140 GHz. A simple theory that accounts for the salient aspects of this pulsed DNP technique is presented. In systems where Overhauser mechanisms dominate the DNP, this new pulsed method can produce enhancements which are comparable to or greater than those obtained with conventional cw irradiation techniques, with lower average microwaves powers.

Bacteriorhodopsin (bR) is a 26 kDa archaeal membrane protein that harvests light energy to create an ion gradient across the cell membrane. Photoisomierzation of the retinilydene chromophore is coupled to ion translocation via a sequence of photocycle intermediates. Here we apply selective multidimensional solid-state NMR to uniformly 13C,15N-labeled bR in its native membrane to obtain chemical shifts in the chromophore of cryogenically trapped bR photointermediates. This is made feasible by using 250 GHz radiation to stimulate dynamic nuclear polarization (DNP), whereby the large spin polarization of unpaired electrons in exogenous free biradicals is transferred to nuclei. Subsequent N-C-C transfers in the NMR experiment allow us to distinguish four discrete substates of the L intermediate. Three of these are shunts that revert to the resting state of the protein upon thermal relaxation, while one L substate, labeled as ‘persistent L’ in our earlier 1D experiments, relaxes to the M state and is therefore deemed functional. Functional L has the strongest counterion, as indicated by its Schiff base (SB) nitrogen chemical shift. It also has a fully planarized 13-cis C13=C14 bond, as indicated by the gamma effect on the C12 chemical shift. These results are consistent with indications from time-resolved optical spectrometry and QM/MM studies of multiple barriers on the way to SB deprotonation. On the other hand, they are inconsistent with models in which the C13=C14 bond is twisted until Schiff base deprotonation. The experiments also demonstrate the use of DNP-enhancement at cryogenic temperatures to investigate mixed states of a membrane protein by multi-dimensional NMR. The results presented here would have been impossible without the availability of DNP to enhance spin polarization that is spread over multiple atoms in multiple protein states.

The theory of hybrid experiments which combine TOSS-type sequences with multiple-pulse-scaling sequences to efficiently eliminate rotational sidebands is described. The analysis relies on the magnetization vector model of magic-angle spinning NMR and leads to a simple derivation of the equations which govern the experiments. New pulse sequences are given for a wide range of experimental conditions.

A series of new two-dimensional dipolar-chemical-shift experiments in magic-anglespinning NMR are presented. A π pulse applied in the center of the evolution period produces dipolar sidebands spaced at one-half the spinning frequency in the ω 1 dimension. The π pulse may be applied to either rare or abundant spins, with differing results upon the spectrum. In both cases the sidebands are more intense than those obtained by conventional methods and should assist the measurement of small heteronuclear dipolar couplings. For one experiment, a dipolar spectrum may be extracted from the 2D landscape which is rigorously identical to the dipolar spectrum at one-half the actual spinning speed. The 2D sideband arrays are sensitive to the relative orientation of the chemical-shift and dipolar tensors, even in the regime where the spinning rate is comparable to the size of one or both tensors.

Low-temperature magic-angle spinning (MAS) NMR experiments are of considerable interest since they permit the study of a variety of phenomena such as chemical exchange, phase transitions, and Knight shifts. In addition, the recent development of techniques for determining distances and distinguishing among molecular conformations in solids provides opportunities to examine the structure of reactions in intermediates (i.e., photolysis products and enzyme/inhibitor or substrate complexes) trapped at low temperatures. In this Note we describe a system that we have developed and successfully employed for performing MAS experiments for extended periods of continuous operation in the temperature range of 150-300 K. The desirable features of a low-temperature MAS system are severalfold. First, it should provide setable and stable spinning speeds for extended periods. For low temperatures, this dictates that at least the drive-bearing gas must be at room temperature and supplied to a spinner-speed controller before cooling in order to maintain constant speeds. Second, the spinning speed must be maintained when the temperature is changed. Third, facilities for replenishing the coolants and the drive and bearing gases must be provided to accommodate operation for extended periods. The first requirement above was initially addressed simply by circulating the drive and bearing gases through copper coils immersed in liquid nitrogen, the temperature being determined by adjusting the size of the coils and the flow rate. This is the method used at present in most laboratories and in most commercial equipment. However, with this arrangement the temperature and spinning speed cannot be adjusted independently. In addition, at the high pressures which are necessary to achieve high spinning speeds, condensation of the nitrogen occurs, resulting in pulsations of the spinning. Kendrick et al. (2) demonstrated that this problem could be circumvented by pressurizing the liquid nitrogen dewar above the pressure of the gas flow. However, this procedure has the disadvantages that it involves a large pressurized vessel (3) subject to strict safety rules in most operating environments, and that it is difficult to replenish the coolant. It is therefore unsatisfactory for samples requiring extended signal averaging periods.

The effects of (−)- Δ 9 -tetrahydrocannabinol ( Δ 9 -THC) on model phospholipid membranes were studied using solid-state 2 H and 13 C nuclear magnetic resonance spectroscopy. Aqueous multilamellar dispersions of dipalmitoylphosphatidylcholine with specific 2 H- and 13 C-labels as endogenous probes at the C 7′ methylene and the carbonyl groups, respectively, of the sn -2 chain were used to study the conformational and dynamic properties of the bilayer as a function of temperature and drug concentration. The drug molecule decreases the phase transition temperature of the bilayer in a concentration dependent manner up to 20 molar percent when full saturation has occurred. The 2 H spectra show that Δ 9 -THC broadens the phase transition during which the spectra acquire a characteristic shape of a two-component system exchanging at an intermediate rate (≈ 10 6 s −1 ) with some liquid crystalline features. Such spectra provide information related to the melting of the phospholipid chains. At intermediate temperatures, the 13 C spectra show a gel-like and a liquid-crystalline-like exchanging components and provide information about a conformational change at the phospholipid glycerol backbone occurring at or near the pretransition. The spectral composition and rate of exchange are both dependent on drug concentration. We have carried out computer simulations of the 13 C spectra and obtained conformational information related to the phase transition process in the bilayer from gel to liquid crystal. Our studies show that Δ 9 -THC has a stronger effect on the sn -2 carbonyl near the bilayer interface than on the lipid chains and serve to describe the membrane perturbing effects of cannabinoids in molecular terms.

The effect of imperfections on the TOSS experiment, used to suppress rotational sidebands in magic-angle-spinning NMR, is examined. We consider the effects of errors in pulse lengths, phases, and timing as well as resonance offset effects. Errors in the phases of the π pulses have no effect on the efficiency of sideband suppression, and only introduce a phase shift into the spectrum. In contrast, errors in the length of the π pulses or in their timing lead to an attenuated center-band as well as the reintroduction of residual sidebands. Our results show that phase alternation of the π pulses does not necessarily compensate for pulse length errors and can, in fact, exacerbate their effects. The use of composite π pulses in TOSS is briefly examined and is shown to yield superior performance for spectral lines far from resonance.

We describe a two-dimensional (2D) magic angle spinning nuclear magnetic resonance experiment which yields a complete separation of isotropic and anisotropic chemical shifts. The experiment relies upon the fact that the evolution of magnetization which occurs under the TOSS sequence can be reversed by the application of a “mirror image” sequence. This leads to a 2D spectrum with isotropic and anisotropic shifts in ω 2 and isotropic shifts alone in ω 1 .

Carbonate ions which replace phosphates—type B—and hydroxide ions—type A—in bone and synthetic apatites were studied by 13C and 1H solid state NMR spectroscopy. Heated apatites and dipolar suppression experiments have been employed to assign the spectra lines. Two overlapping peaks around 170 ppm which behaved differently on heating were assigned to type B carbontes. Pure type A carbonate gave a sharp signal at 166.5 ppm, and we were able to identify this signal in type AB apatite and in a presumably pure type B carbonate apatite. Amorphous calcium phosphate carbonates gave a broad gaussian peak of about 3 ppm at full width half-maximum with a center of gravity at about 168 ppm. The same resonance line, but with lower intensity, was also observed for apatites containing type A and B carbonates. Magic angle spinning 1H NMR spectra of these apatites showed resolved OH− and H2O signals but were inconclusive in identifying specific structural and adsorbed H2O groups. Implications of the NMR data to the crystal structure of carbonate apatites is discussed.