Automated backbone NMR resonance assignment of large proteins using redundant 
linking from a single simultaneous acquisition.

Stanek, J.; Schubeis, T.; Paluch, P.; Güntert, P.; Andreas, L. B.; Pintacuda, G.

doi:10.1021/jacs.0c00251

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Automated backbone NMR resonance assignment of large proteins using redundant linking from a single simultaneous acquisition.

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Andreas, L. B.
Research Group of Solid State NMR Spectroscopy-2, MPI for Biophysical Chemistry, Max Planck Society;

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Citation

Stanek, J., Schubeis, T., Paluch, P., Güntert, P., Andreas, L. B., & Pintacuda, G. (2020). Automated backbone NMR resonance assignment of large proteins using redundant linking from a single simultaneous acquisition. Journal of the American Chemical Society, (in press). doi:10.1021/jacs.0c00251.

Cite as: https://hdl.handle.net/21.11116/0000-0005-C927-D

Abstract

Thanks to magic-angle spinning (MAS) probes with frequencies of 60-100 kHz, the benefit of high sensitivity 1H detection can now be broadly realized in biomolecular solid-state NMR for the analysis of microcrystalline, sedimented, or lipid-embedded preparations. Nonetheless, performing the assignment of all resonances remains a rate-limiting step in protein structural studies, and even the latest optimized protocols fail to perform this step when the protein size exceeds ~20 kDa. Here we leverage the benefits of fast (100 kHz) MAS and high (800 MHz) magnetic fields to design an approach that lifts this limitation. Through the creation, conservation and acquisition of independent magnetization pathways within a single triple-resonance MAS NMR experiment, a single self-consistent dataset can be acquired, providing enhanced sensitivity, reduced vulnerability to machine or sample instabilities, and highly redundant linking that supports fully-automated peak picking and resonance assignment. The method, dubbed RAVASSA (Redundant Assignment Via A Single Simultaneous Acquisition), is demonstrated with the assignment of the largest protein to date in the solid state, the 42.5 kDa maltose binding protein, using a single fully protonated microcrystalline sample and one week of spectrometer time.