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Sci Adv. 2017 Apr 7;3(4):e1601601. doi: 10.1126/sciadv.1601601. eCollection 2017 Apr.

Unraveling self-assembly pathways of the 468-kDa proteolytic machine TET2.

Macek P1,2,3, Kerfah R1,2,3, Boeri Erba E1,2,3, Crublet E1,2,3, Moriscot C1,2,3, Schoehn G1,2,3, Amero C4, Boisbouvier J1,2,3.

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Université Grenoble Alpes, Institut de Biologie Structurale (IBS), Grenoble, France.
CEA, IBS, Grenoble, France.
CNRS, IBS, Grenoble, France.
Centro de Investigaciones Químicas, IICBA, Universidad Autónoma del Estado de Morelos, México.


The spontaneous formation of biological higher-order structures from smaller building blocks, called self-assembly, is a fundamental attribute of life. Although the protein self-assembly is a time-dependent process that occurs at the molecular level, its current understanding originates either from static structures of trapped intermediates or from modeling. Nuclear magnetic resonance (NMR) spectroscopy has the unique ability to monitor structural changes in real time; however, its size limitation and time-resolution constraints remain a challenge when studying the self-assembly of large biological particles. We report the application of methyl-specific isotopic labeling combined with relaxation-optimized NMR spectroscopy to overcome both size- and time-scale limitations. We report for the first time the self-assembly process of a half-megadalton protein complex that was monitored at the structural level, including the characterization of intermediate states, using a mutagenesis-free strategy. NMR was used to obtain individual kinetics data on the different transient intermediates and the formation of final native particle. In addition, complementary time-resolved electron microscopy and native mass spectrometry were used to characterize the low-resolution structures of oligomerization intermediates.


Electron microscopy; Nuclear magnetic resonance; Real-time structural study; Structural Biology; mass spectrometry; quaternary structure; self-assembly

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