Correct protein folding is vital to life, and cells have evolved a wide array of mechanisms to maintain this process. In the cell, protein folding can be initiated co-translationally . As a polypeptide emerges from the ribosome exit tunnel, domains can fold in a sequential manner, minimising the risk of forming misfolded states. The ribosome surface may also play an active role in the co-translational folding process, through interactions with the nascent polypeptide chain (NC) that stabilise folded or unfolded state, and the conformation of the NC on the ribosome may modulate further co-translational processes such as chaperone recruitment or the assembly of quaternary structure. However, the role of the ribosome surface in guiding efficient protein folding is not well understood.
Structural characterisation of emerging NCs is challenging, owing to a combination of limited sample concentration and stability, high molecular weight, and intrinsic flexibility. We have previously developed NMR spectroscopy as a uniquely powerful tool to study the folding of the NC beyond the exit tunnel, and applied this to study the progressive emergence and folding of the FLN5 filamin domain in translationally-arrested ribosome-nascent chain complexes [2, 3].
Here, we continue our investigation of this system with a study of the dynamics of folded FLN5 nascent chains using sensitivity-optimised measurements of relaxation and cross-correlated relaxation in methyl spin systems. We find that the rotational diffusion of the ribosome-tethered domain is greatly restricted relative to the isolated domain in free solution, and that this effect increases at shorter linker lengths, corresponding to earlier stages of translation. From these measurements we can place strong limits on interactions of the domain with the ribosome surface, and therefore the potential chaperone activity of the ribosome surface. In addition, a residue-specific analysis of relaxation reveals anisotropic behaviour, indicating that tethering to the ribosome may bias the orientation of the tethered domain.We will report our progress in determining the perturbed rotational diffusion tensors for such NCs, and their interpretation in terms of interactions with the ribosome surface and the effect of the intervening linker region.
 Waudby, C. A., Dobson, C. M. & Christodoulou, J. Nature and Regulation of Protein Folding on the Ribosome. Trends in Biochemical Sciences 44, 914–926 (2019).
 Cabrita, L. D. et al. A structural ensemble of a ribosome-nascent chain complex during cotranslational protein folding. Nat. Struct. Mol. Biol. 23, 278–285 (2016).
 Cassaignau, A. M. E. et al. A strategy for co-translational folding studies of ribosome-bound nascent chain complexes using NMR spectroscopy. Nat Protoc 11, 1492–1507 (2016).