Burridge, Charles; (2022) The Dynamics of Folded Nascent Chains Studied With NMR Spectroscopy. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Efficient protein folding is vital for biological function. In the cell, this process can begin during biosynthesis, as proteins gradually emerge from the ribosome exit tunnel. Such progressive, co-translational folding of partially synthesised fragments can increase folding efficiency, by reducing the risk of misfolding between adjacent domains. However, the high effective concentration of the ribosome surface itself may potentially also modulate co-translational folding, by forming interactions with the nascent chain which stabilise folded or unfolded states. Solution-state NMR spectroscopy is a uniquely powerful tool to study the conformation and dynamics of the nascent chain beyond the ribosome exit tunnel and probe such effects. The method has previously been applied to characterise the progressive emergence and folding of the FLN5 filamin domain and quantify interactions between the ribosome surface and the unfolded state. However, interactions with folded nascent chains have not so far been studied. As folded states have very different spectroscopic properties to previously studied unfolded states, in this work we sought to develop a toolkit of NMR methods optimised for the analysis of dynamics folded nascent chains. Firstly, we describe the optimisation using mass-spectroscopy of methyl label incorporation into perdeuterated, translationally-arrested ribosome-nascent chain complexes. Secondly, we report the development and application of sensitivityoptimised measurements of relaxation and cross-correlated relaxation in methyl spin systems, which reveal a reduction in the mobility or rotational diffusion of folded FLN5 RNCs. Finally, we combine these approaches with a protein engineering strategy to explore the determinants of folded nascent chain dynamics during translation. We find that mobility increases as translation progresses, but always remains significantly lower than the isolated protein regardless of linker length or composition. We attribute this observation to interactions with the ribosome surface, which we show to be at least partly electrostatic in nature.

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