Protein resonance assignment at MAS frequencies approaching 100 kHz: a quantitative comparison of J-coupling and dipolar-coupling-based transfer methods.
We discuss the optimum experimental conditions to obtain assignment spectra for solid proteins at magic-angle spinning (MAS) frequencies around 100 kHz. 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 93 kHz versus 60 kHz 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 93 kHz MAS. Within 3 days of instrument time, the required spectra were recorded from which the backbone resonances have been assigned to over 96%.
Physical Chemistry, ETH Zürich, Vladimir-Prelog-Weg 2, 8093, Zurich, Switzerland.
Susanne
S
Penzel
Physical Chemistry, ETH Zürich, Vladimir-Prelog-Weg 2, 8093, Zurich, Switzerland.
Albert A
AA
Smith
Physical Chemistry, ETH Zürich, Vladimir-Prelog-Weg 2, 8093, Zurich, Switzerland.
Vipin
V
Agarwal
Physical Chemistry, ETH Zürich, Vladimir-Prelog-Weg 2, 8093, Zurich, Switzerland.
Andreas
A
Hunkeler
NMR Instituut, Tartu Teadus, Tehnomeedikum, Tallinn University of Technology, Akadeemia tee 15a, 19086, Tallinn, Estonia.
Mai-Liis
ML
Org
NMR Instituut, Tartu Teadus, Tehnomeedikum, Tallinn University of Technology, Akadeemia tee 15a, 19086, Tallinn, Estonia. ago.samoson@ttu.ee.
Ago
A
Samoson
Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS/Université de Lyon 1, 7, passage du Vercors, 69367, Lyon, France. a.bockmann@ibcp.fr.
Anja
A
Böckmann
Physical Chemistry, ETH Zürich, Vladimir-Prelog-Weg 2, 8093, Zurich, Switzerland. maer@ethz.ch.
Matthias
M
Ernst
Physical Chemistry, ETH Zürich, Vladimir-Prelog-Weg 2, 8093, Zurich, Switzerland. beme@ethz.ch.
Beat H
BH
Meier
D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry*
D011506:Proteins
Assignment; MAS; Solid-state NMR