Elsevier

Chemical Physics Letters

Volume 635, 16 August 2015, Pages 339-344
Chemical Physics Letters

Improving spectral resolution in biological solid-state NMR using phase-alternated rCW heteronuclear decoupling

https://doi.org/10.1016/j.cplett.2015.07.008Get rights and content

Highlights

  • Efficient heteronuclear dipolar decoupling using phase alternated rCW.

  • Improved spectral resolution in solid-state NMR of biomolecules.

  • Improved robustness towards amplitude/offset of rf irradiation.

  • Improved robustness towards anisotropic nuclear spin interactions.

  • Superior decoupling in solid-state NMR DARR spectra of GB1.

Abstract

The successful application of solid-state NMR spectroscopy for structural study of biological macromolecules requires high spectral resolution. In presence of abundant H1 spins, the resolution of the prevailing C13 or N15 chemical shift encoding experiments critically depends on the availability of efficient and robust heteronuclear decoupling methods in addition to the use of high-field instrumentation and fast sample spinning. Robustness of the decoupling method towards alterations in amplitude/offset of radio frequency fields due to varying sample states is important to ensure recording of spectra with high resolution over long sampling periods for insensitive samples. Here, we present a phase-alternated refocused continuous-wave decoupling method offering better resolution, easier setup, and higher robustness than previous methods. Improved decoupling is in part ascribed to more efficient cancellation of the residual heteronuclear, H1C13, dipolar coupling interactions which are induced by homonuclear, H1H1, dipolar coupling interactions.

Introduction

Solid-state NMR spectroscopy is becoming an increasingly important tool for the atomic scale investigation of biological macromolecules in complex heterogenous environment, including protein complexes, amyloid fibrils, and membrane proteins [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14]. The growing capability of the method is ascribed to development of high-field instrumentation, fast sample spinning probes, new isotope-labelling procedures, and design of efficient pulse sequences for manipulating the spins to ensure high resolution and establishment of structural parameters. The spectral resolution may be improved using higher static magnetic fields through linear scaling of isotropic chemical-shift interactions. This applies to the limit where residual anisotropic nuclear spin interactions become the critical factor, in which case coherent averaging through fast magic-angle spinning (MAS) and radio-frequency (rf) irradiation is needed. In the most typical situation with abundant H1 spins present, it is difficult to record high-resolution H1 spectra implying that spectral resolution is typically achieved through detection of C13 and N15 resonances in C13, N15-isotopically labelled samples. In this case, it is crucial to invoke efficient heteronuclear decoupling to reduce effects from dipolar couplings between the low-γ spin and protons and indirect effects from homonuclear H1H1 interactions such as higher order and cross terms.

With the aim of obtaining high-resolution spectra of low-γ nuclei in solid-state NMR spectroscopy, a great variety of heteronuclear decoupling sequences have been developed over the years which markedly increases the resolution relative to brute-force continuous-wave (CW) decoupling [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32]. These include powerful methods such as two-pulse phase-modulation (TPPM) [17], its supercycled variant SPINAL [20], its frequency-swept variant SWf-TPPM [26], XiX [16], [22], and lately refocussed continuous wave (rCW) [28], [29], [31] decoupling. In general, although overall improving spectral resolution tremendously, such sequences are not straightforward to set up for optimal performance, and often involves optimization of one or more parameters for a given experimental condition. With focus on biological macromolecules in native environment, including mixtures of proteins, lipids, small molecule constituents, water, and salts, it becomes exceedingly difficult to ensure the best decoupling conditions as optimizations on model samples do not necessarily reflect the conditions in the real sample and finding efficient decoupling condition on the sample itself may be highly time consuming or impossible. Furthermore, sample spinning in extended periods of data acquisition may lead to dehydration and susceptibility changes eventually altering the performance of decoupling during measurements. To address these challenges, we here introduce a simple phase-alternated rCW decoupling scheme demonstrating superior decoupling performance, improved tolerance to experimental parameters, and which virtually does not require any parameter optimization for efficient decoupling under varying experimental conditions.

Section snippets

Phase-alternated rCW

Among current heteronuclear decoupling sequences, the rCW schemes [28], [31] appear particularly promising in terms of efficiency, robustness, and ease in setup. Aimed at improving these features, we have here taken the simple rCWA element [31] (Figure 1a) as basis and experimentally optimized the decoupling efficiency through concatenation with a phase-altered variant of this element. The remarkable outcome of this simple operation is seen in the contour plot in Figure 1d showing

Methods: experimental and numerical

The experiments presented here were carried out on a Bruker Avance III wide bore 700 MHz NMR spectrometer (Bruker Biospin, Rheinstetten) and Bruker Avance II wide bore 400 MHz NMR spectrometer (Bruker Biospin, Rheinstetten). All the experiments were performed using a Bruker 2.5 mm XYZ triple-resonance probe. For all the experiments, the initial polarization to C13 was transferred from protons using ramped-amplitude cross polarization (CP) [33].

All simulations were performed using the open source

Conclusions

We conclude that the proposed rCWApA decoupling scheme is a good candidate for heteronuclear dipolar decoupling in biological solid-state NMR in terms of efficiency, ease of setup, and robustness towards experimental parameters. Good decoupling performance virtually requires no optimization for any given sample and even covers a wide range of experimental conditions. Unlike most state-of-the-art decoupling methods, the performance of rCWApA decoupling is also largely unaffected by the presence

Acknowledgements

The project was supported by grants from the Danish National Research Foundation (DNRF59) and the European Commission under the Seventh Framework Programme (FP7), contract Bio-NMR 261863. We thank Dr. Zdenek Tosner for assistance with the SIMSPON implementation.

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