Aromatic Ring Dynamics, Thermal Activation, and Transient Conformations of a 468 kDa Enzyme by Specific 1H–13C Labeling and Fast Magic-Angle Spinning NMR

Autor: Diego F. Gauto, David Gajan, Roman J. Lichtenecker, Alessandro Barducci, Audrey Hessel, Masatsune Kainosho, Paul Schanda, Pavel Macek, Yohei Miyanoiri, Tsutomu Terauchi, Jérôme Boisbouvier, Hugo Pacheco de Freitas Fraga
Rok vydání: 2019
Předmět:
Zdroj: Journal of the American Chemical Society. 141:11183-11195
ISSN: 1520-5126
0002-7863
DOI: 10.1021/jacs.9b04219
Popis: Aromatic residues are located at structurally important sites of many proteins. Probing their interactions and dynamics can provide important functional insight but is challenging in large proteins. Here, we introduce approaches to characterize the dynamics of phenylalanine residues using 1H-detected fast magic-angle spinning (MAS) NMR combined with a tailored isotope-labeling scheme. Our approach yields isolated two-spin systems that are ideally suited for artifact-free dynamics measurements, and allows probing motions effectively without molecular weight limitations. The application to the TET2 enzyme assembly of ∼0.5 MDa size, the currently largest protein assigned by MAS NMR, provides insights into motions occurring on a wide range of time scales (picoseconds to milliseconds). We quantitatively probe ring-flip motions and show the temperature dependence by MAS NMR measurements down to 100 K. Interestingly, favorable line widths are observed down to 100 K, with potential implications for DNP NMR. Furthermore, we report the first 13C R1ρ MAS NMR relaxation-dispersion measurements and detect structural excursions occurring on a microsecond time scale in the entry pore to the catalytic chamber and at a trimer interface that was proposed as the exit pore. We show that the labeling scheme with deuteration at ca. 50 kHz MAS provides superior resolution compared to 100 kHz MAS experiments with protonated, uniformly 13C-labeled samples.
Databáze: OpenAIRE
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