Improving spectral resolution in biological solid-state NMR using phase-alternated rCW heteronuclear decoupling
Graphical abstract
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 spins present, it is difficult to record high-resolution spectra implying that spectral resolution is typically achieved through detection of and resonances in , -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 – 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 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.
References (40)
- et al.
Biophys. J.
(2008) - et al.
J. Mol. Biol.
(2009) - et al.
Cell
(2013) - et al.
J. Magn. Reson. A
(1994) - et al.
Solid State Nucl. Magn. Reson.
(1997) - et al.
J. Magn. Reson.
(2000) - et al.
Chem. Phys. Lett.
(2001) - et al.
Chem. Phys. Lett.
(2002) J. Magn. Reson.
(2003)Prog. Nucl. Magn. Reson. Spectrosc.
(2005)