Forces on Nascent Polypeptides During Membrane Insertion and Translocation via the Sec Translocon

Co-translational insertion into and translocation across the cell membrane via the Sec translocon are key steps in the biogenesis of membrane and secreted proteins. However, study of these processes is challenging due to the role of long-timescale non-equilibrium dynamics during ribosomal translation. We have developed a coarse-grained (CG) simulation approach capable of reaching experimental (i.e. minute) timescales, while retaining sufficient detail to capture the effect of single amino-acid substitutions. The CG model is applied to uncover the mechanism underlying experimentally observed forces acting on hydrophilic, hydrophobic, and charged nascent polypeptides while they are synthesized by the ribosome and pass through the Sec translocon. Calculated forces show strong agreement with experimental force measurements and visualization of the CG trajectories allows us to ascribe observed forces to distinct physical processes acting on the nascent polypeptide. CG simulations with modified interactions confirm these findings and provide experimentally testable hypotheses. This study demonstrates a combined simulation and experimental approach able to provide high-resolution insight into the co-translational integration and translocation of nascent polypeptides.