Maiti, M. | Search bioRxiv

bioRxiv2020

Gradual Compaction of the Nascent Peptide During Cotranslational Folding on the Ribosome

ABSTRACTNascent 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. Folding mutations 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.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2018

Quantitative modelling explains distinct STAT1 and STAT3 activation dynamics in response to both IFNγ and IL-10 stimuli and predicts emergence of reciprocal signalling at the level of single cells

IFN{gamma} and IL10 pathways elicit functionally opposing cell fates, however, these two stimuli activate common transcription factors like STAT1 and STAT3 (S/1/3). How the two stimuli regulate the dynamics of S/1/3 activation remains less understood. Here, we experimentally measured the signaling dynamics of S/1/3 in response to IFN{gamma} and IL10 and found that STAT3, in particular, exhibits a bell-shaped response to both stimuli with maximal activation in an intermediate dose. We built a mathematical model, which quantitatively captured the S/1/3 dynamics and by model analysis, we identified the primary regulators controlling the bell-shaped STAT3 responses in both pathways. As the STATs are activated in response to both stimuli, in a scenario when cells are subjected to co-stimulation of IFN{gamma} and IL10, an interpathway competition to activate the common substrate is plausible. Additionally, a strong transcriptional induction of SOCS1 (a negative regulator of IFN{gamma} pathway) was observed upon IL10 stimulation, suggesting that IL10 pathway can potentially inhibit the IFN{gamma} pathway via SOCS1 induction. To quantitatively understand the S/1/3 responses to co-stimulation we next simulated the same which predicted STAT3 activation and SOCS1 induction dynamics would robustly remain IL10 driven. Subsequent experiments validated the model predictions. Further, the model analysis identified the primary regulators controlling the robustness of IL10-STAT3 axis in co-stimulation. Together, our data-driven model quantitatively captured the dynamics of the STATs in response to both pro (IFN{gamma}) and anti (IL10)-inflammatory stimuli and identifies distinct regulatory elements controlling STAT3 signaling in individual and co-stimulation conditions.

systems biology↗