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ABSTRACT: Resonance assignment is the first step in NMR structure determination. For magic angle spinning NMR, this is typically achieved with a set of heteronuclear correlation experiments (NCaCX, NCOCX, CONCa) that utilize SPECIFIC-CP (15)N-(13)C transfers. However, the SPECIFIC-CP transfer efficiency is often compromised by molecular dynamics and probe performance. Here we show that one-bond ZF-TEDOR (15)N-(13)C transfers provide simultaneous NCO and NCa correlations with at least as much sensitivity as SPECIFIC-CP for some non-crystalline samples. Furthermore, a 3D ZF-TEDOR-CC experiment provides heteronuclear sidechain correlations and robustness with respect to proton decoupling and radiofrequency power instabilities. We demonstrate transfer efficiencies and connectivities by application of 3D ZF-TEDOR-DARR to a model microcrystalline protein, GB1, and a less ideal system, GvpA in intact gas vesicles.
Journal of Biomolecular NMR 01/2013; · 3.61 Impact Factor
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ABSTRACT: We report chemical shift assignments of the drug-resistant S31N mutant of M2(18-60) determined using 3D magic-angle-spinning (MAS) NMR spectra acquired with a (15)N-(13)C ZF-TEDOR transfer followed by (13)C-(13)C mixing by RFDR. The MAS spectra reveal two sets of resonances, indicating that the tetramer assembles as a dimer of dimers, similar to the wild-type channel. Helicies from the two sets of chemical shifts are shown to be in close proximity at residue H37, and the assignments reveal a difference in the helix torsion angles, as predicted by TALOS+, for the key resistance residue N31. In contrast to wild-type M2(18-60), chemical shift changes are minimal upon addition of the inhibitor rimantadine, suggesting that the drug does not bind to S31N M2.
Journal of the American Chemical Society 04/2012; 134(17):7215-8. · 9.91 Impact Factor
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ABSTRACT: We employ a combination of (13)C/(15)N magic angle spinning (MAS) NMR and (2)H NMR to study the structural and functional consequences of different membrane environments on VDAC1 and, conversely, the effect of VDAC1 on the structure of the lipid bilayer. MAS spectra reveal a well-structured VDAC1 in 2D crystals of dimyristoylphosphatidylcholine (DMPC) and diphytanoylphosphatidylcholine (DPhPC), and their temperature dependence suggests that the VDAC structure does not change conformation above and below the lipid phase transition temperature. The same data show that the N-terminus remains structured at both low and high temperatures. Importantly, functional studies based on electrophysiological measurements on these same samples show fully functional channels, even without the presence of Triton X-100 that has been found necessary for in vitro-refolded channels. (2)H solid-state NMR and differential scanning calorimetry were used to investigate the dynamics and phase behavior of the lipids within the VDAC1 2D crystals. (2)H NMR spectra indicate that the presence of protein in DMPC results in a broad lipid phase transition that is shifted from 19 to ~27 °C and show the existence of different lipid populations, consistent with the presence of both annular and bulk lipids in the functionally and structurally homogeneous samples.
Journal of the American Chemical Society 03/2012; 134(14):6375-87. · 9.91 Impact Factor
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ABSTRACT: We show that a simple, general, and easily reproducible method for generating non-uniform sampling (NUS) schedules preserves the benefits of random sampling, including inherently reduced sampling artifacts, while removing the pitfalls associated with choosing an arbitrary seed. Sampling schedules are generated from a discrete cumulative distribution function (CDF) that closely fits the continuous CDF of the desired probability density function. We compare random and deterministic sampling using a Gaussian probability density function applied to 2D HSQC spectra. Data are processed using the previously published method of Spectroscopy by Integration of Frequency and Time domain data (SIFT). NUS spectra from deterministic sampling schedules were found to be at least as good as those from random schedules at the SIFT critical sampling density, and significantly better at half that sampling density. The method can be applied to any probability density function and generalized to greater than two dimensions.
Journal of Magnetic Resonance 12/2011; 214(1):296-301. · 2.14 Impact Factor
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ABSTRACT: We describe magic-angle spinning NMR experiments designed to elucidate the interstrand architecture of amyloid fibrils. Three methods are introduced for this purpose, two being based on the analysis of long-range (13)C-(13)C correlation spectra and the third based on the identification of intermolecular interactions in (13)C-(15)N spectra. We show, in studies of fibrils formed by the 86-residue SH3 domain of PI3 kinase (PI3-SH3 or PI3K-SH3), that efficient (13)C-(13)C correlation spectra display a resonance degeneracy that establishes a parallel, in-register alignment of the proteins in the amyloid fibrils. In addition, this degeneracy can be circumvented to yield direct intermolecular constraints. The (13)C-(13)C experiments are corroborated by (15)N-(13)C correlation spectra obtained from a mixed [(15)N,(12)C]/[(14)N,(13)C] sample which directly quantify interstrand distances. Furthermore, when the spectra are recorded with signal enhancement provided by dynamic nuclear polarization (DNP) at 100 K, we demonstrate a dramatic increase (from 23 to 52) in the number of intermolecular (15)N-(13)C constraints detectable in the spectra. The increase in the information content is due to the enhanced signal intensities and to the fact that dynamic processes, leading to spectral intensity losses, are quenched at low temperatures. Thus, acquisition of low temperature spectra addresses a problem that is frequently encountered in MAS spectra of proteins. In total, the experiments provide 111 intermolecular (13)C-(13)C and (15)N-(13)C constraints that establish that the PI3-SH3 protein strands are aligned in a parallel, in-register arrangement within the amyloid fibril.
Journal of the American Chemical Society 08/2011; 133(35):13967-74. · 9.91 Impact Factor
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Angewandte Chemie International Edition 10/2010; 49(48):9215-8. · 13.45 Impact Factor
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ABSTRACT: The tetrameric M2 proton channel from influenza A virus conducts protons at low pH and is inhibited by aminoadamantyl drugs such as amantadine and rimantadine (Rmt). We report magic angle spinning NMR spectra of POPC and DPhPC membrane-embedded M2(18-60), both apo and in the presence of Rmt. Similar line widths in the spectra of apo and bound M2 indicate that Rmt does not have a significant impact on the dynamics or conformational heterogeneity of this construct. Substantial chemical shift changes for many residues in the transmembrane region support an allosteric mechanism of inhibition. An Rmt titration supports a binding stoichiometry of >1 Rmt molecule per channel and shows that nonspecific binding or changes in membrane composition are unlikely sources of the chemical shift changes. In addition, doubling of spectral lines in all of the observed samples provides evidence that the channel assembles with twofold symmetry.
Journal of the American Chemical Society 08/2010; 132(32):10958-60. · 9.91 Impact Factor
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ABSTRACT: We demonstrate the successful application of (13)C-(13)C proton assisted recoupling (PAR) on [U-(13)C,(15)N] N-f-MLF-OH and [U-(13)C,(15)N] protein GB1 at high magic angle spinning (MAS) frequencies (omega(r)/2pi = 65 kHz). Specifically, by combining PAR mixing with low power heteronuclear decoupling (omega(1H)/2pi approximately 16 kHz) and high spinning frequencies, we obtain high resolution 2D spectra displaying long-range (13)C-(13)C contacts from which distance estimates can be extracted. These experiments therefore demonstrate the possibility of performing high resolution structural studies in the limit of high spinning frequency and low power (1)H decoupling, a regime which optimizes the resolution of protein samples and preserves their integrity.
The Journal of Physical Chemistry B 07/2009; 113(27):9062-9. · 3.70 Impact Factor
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ABSTRACT: Rotational resonance width (R(2)W) magic-angle spinning (MAS) NMR experiments are performed to measure (13)C-(13)C distances in the hydrophobic core of the microcrystalline model protein G(Beta1). Such inter-residue distances are of particular value in NMR structure determinations. The experiments are done at a Larmor frequency of 750 MHz (1)H where the contribution of (13)C chemical shift anisotropy (CSA) to the R(2) transfer mechanism is significant. To minimize line broadening in the 2D spectra, we employ a combination of even/odd isotopic labeling with [1,3-(13)C] glycerol, and J-decoupling in the indirect dimension. This results in high-precision distance measurements between aromatic side chains of three tyrosine residues and distant methyl groups in the hydrophobic core of the protein. Even in the absence of information on the relative orientation of the shift tensors, we obtain relatively high precision data, which can be further improved by additional constraints on the tensor orientations.
ChemPhysChem 07/2009; 10(9-10):1656-63. · 3.41 Impact Factor
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ABSTRACT: We describe a new magic angle spinning (MAS) NMR experiment for obtaining (15)N-(15)N correlation spectra. The approach yields direct information about the secondary and tertiary structure of proteins, including identification of alpha-helical stretches and interstrand connectivity in antiparallel beta-sheets, which are of major interest for structural studies of membrane proteins and amyloid fibrils. The method, (15)N-(15)N proton assisted recoupling (PAR), relies on a second-order mechanism, third spin assisted recoupling (TSAR), used previously in the context of (15)N-(13)C and (13)C-(13)C polarization transfer schemes. In comparison to (15)N-(15)N proton-driven spin diffusion experiments, the PAR technique accelerates polarization transfer between (15)N's by a factor of approximately 10(2)-10(3) and is furthermore applicable over the entire range of currently available MAS frequencies (10-70 kHz).
Journal of the American Chemical Society 05/2009; 131(16):5769-76. · 9.91 Impact Factor
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ABSTRACT: A simple and effective method, SIFT (spectroscopy by integration of frequency and time domain information), is introduced for processing nonuniformly sampled multidimensional NMR data. Applying the computationally efficient Gerchberg-Papoulis (G-P) algorithm, used previously in picture processing and medical imaging, SIFT supplements data at nonuniform points in the time domain with the information carried by known "dark" points (i.e., empty regions) in the frequency domain. We demonstrate that this rapid integration not only removes the severe pseudonoise characteristic of the Fourier transforms of nonuniformly sampled data, but also provides a robust procedure for using frequency information to replace time measurements. The latter can be used to avoid unnecessary sampling in sampling-limited experiments, and the former can be used to take advantage of the ability of nonuniformly sampled data to minimize trade-offs between the signal-to-noise ratio and the resolution in sensitivity-limited experiments. Processing 2D and 3D data sets takes about 0.1 and 2 min, respectively, on a personal computer. With these several attractive features, SIFT offers a novel, model-independent, flexible, and user-friendly tool for efficient and accurate processing of multidimensional NMR data.
Journal of the American Chemical Society 05/2009; 131(13):4648-56. · 9.91 Impact Factor
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ABSTRACT: We describe solid-state NMR homonuclear recoupling experiments at high magic-angle spinning (MAS) frequencies using the radio frequency-driven recoupling (RFDR) scheme. The effect of heteronuclear decoupling interference during RFDR recoupling at high spinning frequencies is investigated experimentally and via numerical simulations, resulting in the identification of optimal decoupling conditions. The effects of MAS frequency, RF field amplitude, bandwidth, and chemical shift offsets are examined. Most significantly, it is shown that broadband homonuclear correlation spectra can be efficiently obtained using RFDR without decoupling during the mixing period in fully protonated samples, thus considerably reducing the rf power requirements for acquisition of (13)C-(13)C correlation spectra. The utility of RFDR sans decoupling is demonstrated with broadband correlation spectra of a peptide and a model protein at high MAS frequencies and high magnetic field.
The Journal of Chemical Physics 03/2008; 128(5):052321. · 3.33 Impact Factor
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ABSTRACT: Several approaches for utilizing dipolar recoupling solid-state NMR (ssNMR) techniques to determine local structure at high resolution in peptides and proteins have been developed. However, many of these techniques measure only one torsion angle or are accurate for only certain classes of secondary structure. Additionally, the efficiency with which these dipolar recoupling experiments suppress the deleterious effects of chemical shift anisotropy (CSA) at high magnetic field strengths varies. Dipolar recoupling with a windowless sequence (DRAWS) has proven to be an effective pulse sequence for exciting double-quantum (DQ) coherences between adjacent carbonyl carbons along the peptide backbone. By allowing this DQ coherence to evolve, it is possible to measure the relative orientations of the CSA tensors and subsequently use this information to determine the Ramachandran torsion angles phi and psi. Here, we explore the accuracies of the assumptions made in interpreting DQ-DRAWS data and demonstrate their fidelity in measuring torsion angles corresponding to a variety of secondary structures irrespective of hydrogen-bonding patterns. It is shown how a simple choice of isotopic labels and experimental conditions allows accurate measurement of backbone secondary structures without any prior knowledge. This approach is considerably more sensitive for determining structure in helices and has comparable accuracy for beta-sheet and extended conformations relative to other methods. We also illustrate the ability of DQ-DRAWS to distinguish between structures in heterogeneous samples.
Journal of the American Chemical Society 03/2008; 130(7):2202-12. · 9.91 Impact Factor
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ABSTRACT: We report solution-state and solid-state 13C NMR data for the alanine dipeptide (N-acetyl-l-alanine-N‘-methylamide, AcAlaNHMe) in polycrystalline, lyophilized, and solvated states. Changes in the 13C chemical shifts of individual carbonyl carbons between the solvated and lyophilized states for the alanine dipeptide dissolved in H2O and CHCl3 suggest preferential solvation at different carbonyl carbons. This effect depends on the solvent, and it suggests an alteration in secondary structure on removal of water. We employ a novel external referencing scheme (neat tetramethylsilane under magic-angle spinning), which allows for a direct comparison of solution- and solid-state chemical shifts. The structure of the alanine dipeptide in the crystalline state and after lyophilization out of H2O and CHCl3 solutions was determined using double-quantum dipolar recoupling solid-state NMR at high magnetic field strength (600 MHz). The compound adopts the same polyproline-II-like secondary structure when lyophilized from hydrogen-bonding (H2O) and non-hydrogen-bonding (CHCl3) solvents.
02/2004;
