Figure 2.

Unfolding kinetics as measured by pulse proteolysis. A, cartoon representation of a ribosome-stalled nascent chain generated for pulse proteolysis experiments. A 50S subunit (orange) and 30S subunit (light blue) with a peptidyl-tRNA (yellow) stalls during translation of mRNA (gray) at a SecM stall sequence (red circles). The protein of interest (green circles) is extended beyond the ribosome exit tunnel by a 10-residue glycine–serine linker (blue circles). The nascent chain is tagged with a BODIPY-FL-Lysine (star). B, representative traces of observed off-ribosome unfolding rates of RNH I53D at 4.5 m urea (black) and 3.0 m urea (gray). C, representative traces of observed on-ribosome unfolding rates of RNH I53D at 2.6 m urea (dark green) and 2.2 m urea (light green). D, data sets including B and C are fit to a single exponential to extract k_obs, and the ln(k_obs) for experiments on (green) and off the ribosome (gray) are plotted at several urea concentrations. Error bars are the 95% confidence interval of the fits to a single exponential at each urea concentration. These data are fit to a linear model weighted for the error at each point where the slope is $m_{\text{unf}}^{‡}$, and the y intercept is $k_{\text{unf}}^{\text{0M urea}}$, the unfolding rate in a 0 m denaturant condition. The urea concentrations at which we can investigate the unfolding rates of nascent chains are limited based on the C_m of the nascent chain and the stability of 70S ribosomes, leading to a higher error for the extrapolation of on-ribosome data.