Partition Coefficient of Arginine between Translocon Interior and Lipid Phase

Abstract

Depending on its hydrophobicity, the protein translocation complex sorts polypeptide segments into the membrane or into the aqueous solution. Its short residence time in the channel limits polypeptide sampling between the aqueous lumen and the membrane interior, giving rise to an out-of-equilibrium condition. Moreover, reduced mobility and orientational restrictions of confined intraluminal water molecules are bound to further bias peptide partition into the membrane, rendering good agreement between the physico-chemical and biological hydrophobicity scales all the more surprising. Here we report the equilibrium partition coefficient of arginine derived on the single molecule level by monitoring -repetitive lateral movement of a stalled polypeptide segment between the SecYEG lumen and the lipid interior. Polypeptide movements in the direction normal to the membrane were inhibited by sandwiching the transmembrane sequence between a SecA binding sequence and the sequence of the calmodulin binding protein. With calmodulin and SecA bound to opposite sides of the membrane, polypeptide residence within the SecYEG pore was indicated by channel opening, whereas episodes of residence in the hydrophobic membrane interior corresponded to channel closings. Translocon purification and reconstitution into planar lipid bilayers were performed as described previously.1-2 Comparison to amino acid partitioning in a biphasic system (physico-chemical hydrophobicity scale) allowed us to address the effects of water confinement by the SecYEG pore, while comparison to the biological hydrophobicity scale reveals out-of-equilibrium effects. 1. Knyazev, D. G.; ⋯; Pohl, P., The bacterial translocon SecYEG opens upon ribosome binding. J. Biol. Chem. 2013, 288 (25), 17941-6. 2. Knyazev, D. G.;⋯ ; Pohl, P., Ion conductivity of the bacterial translocation channel SecYEG engaged in translocation. J. Biol. Chem. 2014, 289 (35), 24611-6.