Wentink, AS; (2015) Structure and Dynamics of the E. coli Chaperone, Trigger Factor, and its Interaction with Ribosome-Nascent Chain Complexes. Doctoral thesis , UCL (University College London).

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Abstract

The de novo folding of newly synthesised polypeptide chains, exposed in a vectorial manner to the crowded cellular environment, often requires the assistance of molecular chaperones. The ribosome-associated molecular chaperone, trigger factor (TF), facilitates the early folding events of such nascent chains at the ribosomal exit tunnel. The investigation of the structural and dynamic properties of this interaction, at high resolution, presents a challenge due to the dynamic nature of the interaction and the intrinsic conformational heterogeneity of nascent chains. Additional complexity comes from the competition between TF self-association into a dimeric state, the interaction with ribosome-nascent chain complexes (RNCs) and the interaction with isolated protein substrates independent of the ribosome. The sensitivity of NMR spectroscopy to both structural and dynamic changes make this technique uniquely suited to the investigation of the trigger factor chaperone. This thesis presents the application of selective isotopic labelling strategies and advanced TROSY optimised NMR experiments to the study of the TF dimerisation equilibrium, providing a detailed understanding of its kinetics and thermodynamics and insights into the solution structure of the TF dimer. The improved understanding of the mechanism of TF dimerisation forms the basis for the interpretation of observations made in the presence of substrate proteins. Preliminary results on the interaction of TF with two disordered model ribosome- nascent chain complexes reveal that ribosome-associated nascent chain substrates are significantly perturbed by the presence of TF, even in the absence of predicted TF binding motifs. The study of TF and the corresponding isolated polypeptides reveals that limited interaction occurs in the absence of the ribosome. This early NMR investigation of the TF chaperone provides the first residue-specific details of the highly dynamic interaction of TF with nascent polypeptides and begins to elucidate the role of this chaperone in the process of co-translational folding.

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