Forces on nascent polypeptides during membrane insertion and translocation via the Sec translocon

Abstract

Co-translational insertion into and translocation across the cell membrane via the Sec translocon are key steps in the biogenesis of membrane proteins and secreted proteins. However, both exptl. and computational studies of these processes are difficult due to the role of long-timescale non-equil. dynamics during ribosomal translation. We have developed a coarsegrained (CG) simulation approach that is capable of reaching exptl. relevant (i.e. minute) timescales, while retaining the level of detail required to capture the effect of individual amino-acid substitutions. The CG model is applied to uncover the mechanism underlying exptl. obsd. forces acting on hydrophilic, hydrophobic, and charged nascent polypeptides as they are synthesized by the ribosome and pass through the Sec translocon. Forces calcd. using the CG model show excellent agreement with exptl. force measurements using arrest peptide assays. Visualization of the resulting CG trajectories allows us to ascribe obsd. forces to distinct phys. processes acting on the nascent polypeptide, and addnl. CG simulations with modified interactions are used to confirm these findings and provide exptl. testable hypotheses. This study demonstrates the exciting potential of a combined simulation and exptl. approach to provide high-resoln. insight into key phys. processes that take place during the co-translational integration and translocation of nascent polypeptides.