Abstract
The driving force behind protein translocation across the cell membrane is not yet fully understood. In bacteria, there is an electrochemical potential across the cell membrane, which can interact with charged residues in the translocation substrate. In this study, the protonation states of lysine and glutamate, serving as test residues in a peptide translocating across the bacterial channel SecYEG, are investigated by applying Poisson-Boltzmann continuum electrostatic free energy calculations and Monte Carlo titrations to snapshots of molecular dynamics (MD) simulations. A clear shift in protonation probability towards the uncharged state is found for both test residues as they move deeper into the channel. Thus, charge neutralization occurs irrespective of whether the original charge of the test residue is positive (lysine) or negative (glutamate). Electrostatic interactions of acidic and basic residues of SecYEG with the peptide cancel out. The main determinants of the test residue's protonation state are the dielectric properties of its surroundings and interactions with non-titrating charges in the channel. Crucially, the membrane protein-including its water-filled pore-is assigned a low dielectric constant. The results are discussed in the context of the limitations inherent to continuum electrostatics and MD simulations with fixed protonation states.