Free-energy cost for translocon-assisted insertion of membrane proteins

Nascent membrane proteins typically insert in a sequential fashion into the membrane via a protein-conducting channel, the Sec translocon. How this process occurs is still unclear, although a thermodynamic partitioning between the channel and the membrane environment has been proposed. Experiment-and simulationbased scales for the insertion free energy of various amino acids are, however, at variance, the former appearing to lie in a narrower range than the latter. Membrane insertion of arginine, for instance, requires 14-17 kcal/mol according to molecular dynamics simulations, but only 2-3 kcal/mol according to experiment. We suggest that this disagreement is resolved by assuming a two-stage insertion process wherein the first step, the insertion into the translocon, is energized by protein synthesis and, therefore, has an effectively zero free-energy cost; the second step, the insertion into the membrane, invokes the translocon as an intermediary between the fully hydrated and the fully inserted locations. Using free-energy perturbation calculations, the effective transfer free energies from the translocon to the membrane have been determined for both arginine and leucine amino acids carried by a background polyleucine helix. Indeed, the insertion penalty for arginine as well as the insertion gain for leucine from the translocon to the membrane is found to be significantly reduced compared to direct insertion from water, resulting in the same compression as observed in the experiment-based scale.