Protein resonance assignment at MAS frequencies approaching 100kHz: a quantitative comparison of J-coupling and dipolar-coupling-based transfer methods
Penzel, Susanne ; Smith, Albert ; Agarwal, Vipin ; Hunkeler, Andreas ; Org, Mai-Liis ; Samoson, Ago ; Böckmann, Anja ; Ernst, Matthias ; Meier, Beat
In: Journal of Biomolecular NMR, 2015, vol. 63, no. 2, p. 165-186
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- We discuss the optimum experimental conditions to obtain assignment spectra for solid proteins at magic-angle spinning (MAS) frequencies around 100kHz. We present a systematic examination of the MAS dependence of the amide proton T 2′ times and a site-specific comparison of T 2′ at 93kHz versus 60kHz MAS frequency. A quantitative analysis of transfer efficiencies of building blocks, as they are used for typical 3D experiments, was performed. To do this, we compared dipolar-coupling and J-coupling based transfer steps. The building blocks were then combined into 3D experiments for sequential resonance assignment, where we evaluated signal-to-noise ratio and information content of the different 3D spectra in order to identify the best assignment strategy. Based on this comparison, six experiments were selected to optimally assign the model protein ubiquitin, solely using spectra acquired at 93kHz MAS. Within 3days of instrument time, the required spectra were recorded from which the backbone resonances have been assigned to over 96%.