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Spin diffusion driven by R-symmetry sequences: applications to homonuclear correlation spectroscopy in MAS NMR of biological and organic solids.

ABSTRACT: We present a family of homonuclear (13)C-(13)C magic angle spinning spin diffusion experiments, based on R2(n)(v) (n = 1 and 2, v = 1 and 2) symmetry sequences. These experiments are well suited for (13)C-(13)C correlation spectroscopy in biological and organic systems and are especially advantageous at very fast MAS conditions, where conventional PDSD and DARR experiments fail. At very fast MAS frequencies the R2(1)(1), R2(2)(1), and R2(2)(2) sequences result in excellent quality correlation spectra both in model compounds and in proteins. Under these conditions, individual R2(n)(v) display different polarization transfer efficiency dependencies on isotropic chemical shift differences: R2(2)(1) recouples efficiently both small and large chemical shift differences (in proteins these correspond to aliphatic-to-aliphatic and carbonyl-to-aliphatic correlations, respectively), while R2(1)(1) and R2(2)(2) exhibit the maximum recoupling efficiency for the aliphatic-to-aliphatic or carbonyl-to-aliphatic correlations, respectively. At moderate MAS frequencies (10-20 kHz), all R2(n)(v) sequences introduced in this work display similar transfer efficiencies, and their performance is very similar to that of PDSD and DARR. Polarization transfer dynamics and chemical shift dependencies of these R2-driven spin diffusion (RDSD) schemes are experimentally evaluated and investigated by numerical simulations for [U-(13)C,(15)N]-alanine and the [U-(13)C,(15)N] N-formyl-Met-Leu-Phe (MLF) tripeptide. Further applications of this approach are illustrated for several proteins: spherical assemblies of HIV-1 U-(13)C,(15)N CA protein, U-(13)C,(15)N-enriched dynein light chain DLC8, and sparsely (13)C/uniformly (15)N enriched CAP-Gly domain of dynactin. Due to the excellent performance and ease of implementation, the presented R2(n)(v) symmetry sequences are expected to be of wide applicability in studies of proteins and protein assemblies as well as other organic solids by MAS NMR spectroscopy.

SUBMITTER: Hou G

PROVIDER: S-EPMC3148607 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Spin diffusion driven by R-symmetry sequences: applications to homonuclear correlation spectroscopy in MAS NMR of biological and organic solids.

Hou Guangjin G Yan Si S Sun Shangjin S Han Yun Y Byeon In-Ja L IJ Ahn Jinwoo J Concel Jason J Samoson Ago A Gronenborn Angela M AM Polenova Tatyana T

Journal of the American Chemical Society 20110301 11

We present a family of homonuclear (13)C-(13)C magic angle spinning spin diffusion experiments, based on R2(n)(v) (n = 1 and 2, v = 1 and 2) symmetry sequences. These experiments are well suited for (13)C-(13)C correlation spectroscopy in biological and organic systems and are especially advantageous at very fast MAS conditions, where conventional PDSD and DARR experiments fail. At very fast MAS frequencies the R2(1)(1), R2(2)(1), and R2(2)(2) sequences result in excellent quality correlation sp ...[more]

PMID: 21361320

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Project description:Fast magic angle spinning (MAS) NMR spectroscopy is emerging as an essential analytical and structural biology technique. Large resolution and sensitivity enhancements observed under fast MAS conditions enable structural and dynamics analysis of challenging systems, such as large macromolecular assemblies and isotopically dilute samples, using only a fraction of material required for conventional experiments. Homonuclear dipolar-based correlation spectroscopy constitutes a centerpiece in the MAS NMR methodological toolbox, and is used essentially in every biological and organic system for deriving resonance assignments and distance restraints information necessary for structural analysis. Under fast MAS conditions (rotation frequencies above 35-40 kHz), dipolar-based techniques that yield multi-bond correlations and non-trivial distance information are ineffective and suffer from low polarization transfer efficiency. To overcome this limitation, we have developed a family of experiments, CORD-RFDR. These experiments exploit the advantages of both zero-quantum RFDR and spin-diffusion based CORD methods, and exhibit highly efficient and broadband dipolar recoupling across the entire spectrum, for both short-range and long-range correlations. We have verified the performance of the CORD-RFDR sequences experimentally on a U-(13)C,(15)N-MLF tripeptide and by numerical simulations. We demonstrate applications of 2D CORD-RFDR correlation spectroscopy in dynein light chain LC8 and HIV-1 CA tubular assemblies. In the CORD-RFDR spectra of LC8 acquired at the MAS frequency of 40 kHz, many new intra- and inter-residue correlations are detected, which were not observed with conventional dipolar recoupling sequences. At a moderate MAS frequency of 14 kHz, the CORD-RFDR experiment exhibits excellent performance as well, as demonstrated in the HIV-1 CA tubular assemblies. Taken together, the results indicate that CORD-RFDR experiment is beneficial in a broad range of conditions, including both high and moderate MAS frequencies and magnetic fields.

Project description:We recently described a family of experiments for R2n(v) Driven Spin Diffusion (RDSD) spectroscopy suitable for homonuclear correlation experiments under fast MAS conditions [G. Hou, S. Yan, S.J. Sun, Y. Han, I.J. Byeon, J. Ahn, J. Concel, A. Samoson, A.M. Gronenborn, T. Polenova, Spin diffusion drive by R-symmetry sequencs: applications to homonuclear correlation spectroscopy in MAS NMR of biological and organic solids, J. Am. Chem. Soc. 133 (2011) 3943-3953]. In these RDSD experiments, since the broadened second-order rotational resonance conditions are dominated by the radio frequency field strength and the phase shifts, as well as the size of reintroduced dipolar couplings, the different R2n(v) sequences display unique polarization transfer behaviors and different recoupling frequency bandwidths. Herein, we present a series of modified R2n(v) sequences, dubbed COmbined R2n(v)-Driven (CORD), that yield broadband homonuclear dipolar recoupling and give rise to uniform distribution of cross peak intensities across the entire correlation spectrum. We report NMR experiments and numerical simulations demonstrating that these CORD spin diffusion sequences are suitable for broadband recoupling at a wide range of magnetic fields and MAS frequencies, including fast-MAS conditions (?r=40 kHz and above). Since these CORD sequences are largely insensitive to dipolar truncation, they are well suited for the determination of long-range distance constraints, which are indispensable for the structural characterization of a broad range of systems. Using U-(13)C,(15)N-alanine and U-(13)C,(15)N-histidine, we show that under fast-MAS conditions, the CORD sequences display polarization transfer efficiencies within broadband frequency regions that are generally higher than those offered by other existing spin diffusion pulse schemes. A 89-residue U-(13)C,(15)N-dynein light chain (LC8) protein has also been used to demonstrate that the CORD sequences exhibit uniformly high cross peak intensities across the entire chemical shift range.

Project description:The Lee-Goldburg condition for homonuclear decoupling in 1H magic-angle spinning (MAS) solid-state NMR sets the angle θ, corresponding to arctan of the ratio of the rf nutation frequency, ν1, to the rf offset, to be the magic angle, θm, equal to tan-1(√2) = 54.7°. At 60 kHz MAS, we report enhanced decoupling compared to MAS alone in a 1H spectrum of 15N-glycine with at θ = 30° for a ν1 of ∼100 kHz at a 1H Larmor frequency, ν0, of 500 MHz and 1 GHz, corresponding to a high chemical shift scaling factor (λCS) of 0.82. At 1 GHz, we also demonstrate enhanced decoupling compared to 60 kHz MAS alone for a lower ν1 of 51 kHz, i.e., a case where the nutation frequency is less than the MAS frequency, with θ = 18°, λCS = 0.92. The ratio of the rotor period to the decoupling cycle time, Ψ = τr/τc, is in the range 0.53 to 0.61. Windowed decoupling using the optimised parameters for a ν1 of ∼100 kHz also gives good performance in a 1H spin-echo experiment, enabling implementation in a 1H-detected 15N-1H cross polarisation (CP)-refocused INEPT heteronuclear correlation NMR experiment. Specifically, initial 15N transverse magnetisation as generated by 1H-15N CP is transferred back to 1H using a refocused INEPT pulse sequence employing windowed 1H decoupling. Such an approach ensures the observation of through-bond N-H connectivities. For 15N-glycine, while the CP-refocused INEPT experiment has a lower sensitivity (∼50%) as compared to a double CP experiment (with a 200 μs 15N to 1H CP contact time), there is selectivity for the directly bonded NH3+ moiety, while intensity is observed for the CH21H resonances in the double CP experiment. Two-dimensional 15N-1H correlation MAS NMR spectra are presented for the dipeptide β-AspAla and the pharmaceutical cimetidine at 60 kHz MAS, both at natural isotopic abundance. For the dipeptide β-AspAla, different build-up dependence on the first spin-echo duration is observed for the NH and NH3+ moieties demonstrating that the experiment could be used to distinguish resonances for different NHx groups.

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Spin diffusion driven by R-symmetry sequences: applications to homonuclear correlation spectroscopy in MAS NMR of biological and organic solids.

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Spin diffusion driven by R-symmetry sequences: applications to homonuclear correlation spectroscopy in MAS NMR of biological and organic solids.

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