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Atomic-resolution studies of structure, dynamics and interactions in chaperone assemblies by NMR spectroscopy.

Abstract : The diverse group of molecular chaperones is dedicated to accompany, fold and protect other proteins until they reach their final conformation and loca- tion inside the cell. To this end, molecular chaperones need to be specialized in performing specific tasks, like folding, transport or disaggregation, and versatile in their recognition pattern to engage many di erent client pro- teins. Moreover, molecular chaperones need to be able to interact with each other and with other components of the protein quality control system in a complex network. Interactions between the di erent partners in this network and between the substrate and the chaperone are often dynamic processes, which are especially di cult to study using standard structural biology tech- niques. Consequently, structural data on chaperone/substrate complexes are sparse, and the mechanisms of chaperone action are poorly understood. In this thesis I present investigations of the structure, dynamics and substrate- interactions of two molecular chaperones, using various biophysical and in vivo methods.In the first part I show that the mitochondrial membrane protein chap- erone TIM910 binds its substrates in a highly dynamic manner. Not only is the TIM910 complex in constant exchange between monomeric and hex- americ species, but also the bound substrate samples multiple conformations on a millisecond timescale. Based on nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS), analytical ultracentrifugation (AUC) and in vivo mutational experiments I propose a structural model of the chap- erone/membrane protein interaction. TIM910 binds its substrates in a hy- drophobic pocket on the exterior of the chaperone in a modular fashion, where the number of TIM910 complexes bound depends on the length of the substrate.In the second part I studied the behavior of the N-terminal receptor do- main of the ClpC1 unfoldase from M.tuberculosis in the presence of di erent antibiotics and ligands. The N-terminal domain of ClpC1 is the binding site for various new antibiotics against M.tuberculosis. The antibiotic cyclomarin completely abolishes dynamics induced by the ligand arginine-phosphate. We propose that this suppression of dynamics is the underlying principle for the mechanism of action of this antibiotic.In both cases X-ray structures of the apo or antibiotic bound form were available, but not su cient to explain the mechanism of action. The X- ray structure of TIM910 provided no evidence on where or how substrates are bound. Likewise, X-ray structures of the apo and cyclomarin-bound N-terminal domain of ClpC1 show only minor di erences in structure.Both examples show that static structural data is often not enough to explain how a molecular system works, and only the combination of di er- ent techniques, including newly developed methods enable the atomic-level understanding of chaperone/substrate complexes.
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Submitted on : Friday, July 23, 2021 - 4:34:10 PM
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Katharina Weinhaeupl. Atomic-resolution studies of structure, dynamics and interactions in chaperone assemblies by NMR spectroscopy.. Agricultural sciences. Université Grenoble Alpes, 2018. English. ⟨NNT : 2018GREAV002⟩. ⟨tel-03298518⟩



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