Wspólnotowy Serwis Informacyjny Badan i Rozwoju - CORDIS


SEENANOLIFEINACTION Streszczenie raportu

Project ID: 260887
Źródło dofinansowania: FP7-IDEAS-ERC
Kraj: France

Final Report Summary - SEENANOLIFEINACTION (Real-Time Studies of Biological NanoMachines in Action by NMR)

The study of the assembling, structural and functional properties of biomolecular nanomachines remains a considerable practical challenge. The sheer size of these nanoparticles, the complexity of the structural rearrangements involved present an array of logistical problems. Even if X-ray crystallography and cryo-EM methods can provide static pictures of the system, kinetic data are necessary for a full, atomic resolution understanding of the mode of action.

NMR spectroscopy offers an unique ability to monitor structural and dynamic changes in real-time and at atomic resolution. However, the NMR studies of large proteins and complexes has been a real challenge for a long time. Recent developments in specific isotope labeling of methyl groups in a perdeuterated protein has significantly extended the frontier of liquid state NMR. In recent years, we have exploited metabolic pathways in E. coli and synthesized new isotope-labelled precursors to allow the labeling of any combination of methyl groups in proteins reporting directly on the structure and dynamics of both the protein backbone and side chains extremities.
Using both of these approaches, we have demonstrated that high quality 2D [1H,13C]-methyl spectra of a half MegaDalton protein assembly were acquired in 1 s. We applied these approaches to study in real-time the self-assembly process of the 468 kDa dodecameric tetrahedral aminopeptidase 2 from Pyrococcus horikoshii (TET2). During the self-assembly, we were able to detect signals of two different intermediates. In combination with acquired electron microscopy snapshots and native mass-spectrometry we established that the transient intermediates reflect different oligomerization states in the assembly pathway.

Theses approaches have also been used to probe different functional states and refolding cycle of a 1 MDa active chaperonin. To decipher this mechanism, we have reconstituted the functional assembly specifically labeled on methionine methyl groups. An approach based on mutagenesis of methyl groups residue has been used to assign NMR signals of the methionine residues. Thereby methionine residues have been use as probes of the chaperonin structure allowing the identification of NMR spectra corresponding to the intermediate state and active species of the functional cycle. NMR allowed us to investigate in an atomic- and time-resolved manner the structural rearrangement corresponding to the different states during the functional cycle of a large biological machinery processing its substrate.

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