Reconciling the Roles of Kinetic and Thermodynamic Factors in Membrane–Protein Insertion

[Image: see text] For the vast majority of membrane proteins, insertion into a membrane is not direct, but rather is catalyzed by a protein-conducting channel, the translocon. This channel provides a lateral exit into the bilayer while simultaneously offering a pathway into the aqueous lumen. The determinants of a nascent protein’s choice between these two pathways are not comprehensively understood, although both energetic and kinetic factors have been observed. To elucidate the specific roles of some of these factors, we have carried out extensive all-atom molecular dynamics simulations of different nascent transmembrane segments embedded in a ribosome-bound bacterial translocon, SecY. Simulations on the μs time scale reveal a spontaneous motion of the substrate segment into the membrane or back into the channel, depending on its hydrophobicity. Potential of mean force (PMF) calculations confirm that the observed motion is the result of local free-energy differences between channel and membrane. Based on these and other PMFs, the time-dependent probability of membrane insertion is determined and is shown to mimic a two-state partition scheme with an apparent free energy that is compressed relative to the molecular-level PMFs. It is concluded that insertion kinetics underlies the experimentally observed thermodynamic partitioning process.