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

During ribosomal translation, nascent polypeptide chains (NCs) undergo a variety of physical processes that determine their fate in the cell. This study utilizes a combination of arrest peptide (AP) experiments and coarse-grained molecular dynamics (CGMD) to measure and elucidate the molecular origins of forces that are exerted on NCs during co-translational membrane insertion and translocation via the Sec translocon. The approach enables deconvolution of force contributions from NC-translocon and NC-ribosome interactions, membrane partitioning, and electrostatic coupling to the membrane potential. In particular, we show that forces due to NC-lipid interactions provide a read-out of conformational changes in the Sec translocon, demonstrating that lateral gate opening only occurs when a sufficiently hydrophobic segment of NC residues reaches the translocon. The combination of experiment and theory introduced here provides a detailed picture of the molecular interactions and conformational changes during ribosomal translation that govern protein biogenesis