Abstract
The folding of many proteins can begin during biosynthesis on the ribosome and can be modulated by the ribosome itself. Such perturbations are generally believed to be mediated through interactions between the nascent chain and the ribosome surface, but despite recent progress in characterising interactions of unfolded states with the ribosome, and their impact on the initiation of co-translational folding, a complete quantitative analysis of interactions across both folded and unfolded states of a nascent chain has yet to be realised. Here we apply solution-state NMR spectroscopy to measure transverse proton relaxation rates for methyl groups in folded ribosome–nascent chain complexes of the FLN5 filamin domain. We observe substantial increases in relaxation rates for the nascent chain relative to the isolated domain, which can be related to changes in effective rotational correlation times using measurements of relaxation and cross-correlated relaxation in the isolated domain. Using this approach, we can identify interactions between the nascent chain and the ribosome surface, driven predominantly by electrostatics, and by measuring the change in these interactions as the subsequent FLN6 domain emerges, we may deduce their impact on the free energy landscapes associated with the co-translational folding process.
Highlights
IntroductionCo-translational folding is a fundamental mechanism for ensuring that nascent polypeptide chains (NCs) efficiently acquire and assemble their correct tertiary and quaternary structures following biosynthesis by the ribosome.[1,2] It is increasingly apparent that the ribosome itself can play a role in regulating or modulating this process,[3] and interactions between NCs and the nearby ribosomal surface have been suggested to provide a simple mechanism through which this may be achieved.[4,5,6] the direct measurement of such intramolecular interactions, involving highly dynamic regions of a 2.3 MDa complex, presents a formidable experimental challenge that has only recently begun to be met.[7]We have previously studied the co-translational folding of FLN5, the h immunoglobulin domain from the tandem repeat protein lamin, using the SecM arrest peptide to generate translationally-arrested RNCs that are tethered to the peptidyl-transferase center by varying lengths of the subsequent FLN6 domain[8,9] (Fig. 1A)
We have previously studied the co-translational folding of FLN5, the h immunoglobulin domain from the tandem repeat protein lamin, using the SecM arrest peptide to generate translationally-arrested RNCs that are tethered to the peptidyl-transferase center by varying lengths of the subsequent FLN6 domain[8,9] (Fig. 1A)
The resulting 1H,13C correlation spectrum is shown in Fig. 1C, and from this approximately twenty FLN5 resonances can be resolved unambiguously
Summary
Co-translational folding is a fundamental mechanism for ensuring that nascent polypeptide chains (NCs) efficiently acquire and assemble their correct tertiary and quaternary structures following biosynthesis by the ribosome.[1,2] It is increasingly apparent that the ribosome itself can play a role in regulating or modulating this process,[3] and interactions between NCs and the nearby ribosomal surface have been suggested to provide a simple mechanism through which this may be achieved.[4,5,6] the direct measurement of such intramolecular interactions, involving highly dynamic regions of a 2.3 MDa complex, presents a formidable experimental challenge that has only recently begun to be met.[7]We have previously studied the co-translational folding of FLN5, the h immunoglobulin domain from the tandem repeat protein lamin, using the SecM arrest peptide to generate translationally-arrested RNCs that are tethered to the peptidyl-transferase center by varying lengths of the subsequent FLN6 domain[8,9] (Fig. 1A). Co-translational folding is a fundamental mechanism for ensuring that nascent polypeptide chains (NCs) efficiently acquire and assemble their correct tertiary and quaternary structures following biosynthesis by the ribosome.[1,2] It is increasingly apparent that the ribosome itself can play a role in regulating or modulating this process,[3] and interactions between NCs and the nearby ribosomal surface have been suggested to provide a simple mechanism through which this may be achieved.[4,5,6] the direct measurement of such intramolecular interactions, involving highly dynamic regions of a 2.3 MDa complex, presents a formidable experimental challenge that has only recently begun to be met.[7]. 7) with a systematic survey of dynamics within folded states An understanding of the dynamics and interactions of both folded and unfolded species is essential to understand the co-translational folding equilibrium fully,[3] and so in this work we complement measurements of interactions of the unfolded state of FLN5 (ref. 7) with a systematic survey of dynamics within folded states
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