Abstract
Nascent polypeptides begin to fold in the constrained space of the ribosomal peptide exit tunnel. Here we use force-profile analysis (FPA) and photo-induced energy-transfer fluorescence correlation spectroscopy (PET-FCS) to show how a small α-helical domain, the N-terminal domain of HemK, folds cotranslationally. Compaction starts vectorially as soon as the first α-helical segments are synthesized. As nascent chain grows, emerging helical segments dock onto each other and continue to rearrange at the vicinity of the ribosome. Inside or in the proximity of the ribosome, the nascent peptide undergoes structural fluctuations on the µs time scale. The fluctuations slow down as the domain moves away from the ribosome. Mutations that destabilize the packing of the domain's hydrophobic core have little effect on folding within the exit tunnel, but abolish the final domain stabilization. The results show the power of FPA and PET-FCS in solving the trajectory of cotranslational protein folding and in characterizing the dynamic properties of folding intermediates.
Highlights
The ribosome synthesizes proteins according to the sequence of the messenger RNA by progressively adding amino acids to the C-terminus of the nascent peptide
Previous time-resolved experiments showed that the observed folding dynamics is rapid compared to the pace of translation, and that the nascent chains arrested at different stages of translation faithfully reflect the dynamics that occurs during synthesis[6]
We generated a construct that encodes aa 1 to 101 of HemK, which includes the N-terminal domain (NTD) and part of the interdomain linker connecting the NTD to the C-terminal domain, followed by 17 codons for the SecM arrest peptide, and an additional sequence encoding 20 aa of protein CspA; the latter served as a reporter for high-tension events in the nascent chain
Summary
The ribosome synthesizes proteins according to the sequence of the messenger RNA (mRNA) by progressively adding amino acids to the C-terminus of the nascent peptide. A high-tension mechanical pulling force (7 pN on average) originating from nascent protein folding[29], can alleviate the SecM stalling and restart translation[14,26,29] This phenomenon is utilized in arrest peptide-mediated force profile assays (FPA) to monitor force generation events at various stages of cotranslational folding[14,24,26,29,30,31]. Using PET-FCS to monitor cotranslational folding intermediates on the ribosome could provide insight into their structural dynamics and detect rapid local fluctuations between different conformations of the nascent chain, but so far such experiments have not been carried out. We identify the timing of folding events for the wild-type (wt) NTD and its destabilized 4xA variant, uncover the rates of conformational fluctuations of cotranslational folding intermediates, and define the contribution of the ribosome in maintaining the stability of these compact structures
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