ABSTRACT
The OccK protein subfamily located in the outer membrane of Pseudomonas aeruginosa contains dynamic channels with several conformational states that range from open to closed forms. The molecular determinants of the OccK channels that contribute to the diverse gating have, however, remained elusive so far. Performing molecular dynamics (MD) simulations on OccK5 (OpdH) as an example, local fluctuations of loop L7 mediated by a single residue were identified that effectively gate the channel. The features of this gate residue were studied by single-channel electrophysiology and site-directed mutagenesis demonstrating that this gate residue indeed confers unique gating properties to the OccK channels. In support of these functional measurements, MD simulations highlight the correlations between the size of the side-chain belonging to the gate residue on one side and the pore size as well as the L7 flexibility on the other side.
Subject(s)
Bacterial Outer Membrane Proteins/metabolism , Ion Channels/metabolism , Pseudomonas aeruginosa/metabolism , Amino Acid Sequence , Bacterial Outer Membrane Proteins/chemistry , Humans , Ion Channel Gating , Ion Channels/chemistry , Models, Molecular , Molecular Dynamics Simulation , Protein Conformation , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/chemistryABSTRACT
Single channel electrophysiological studies have been carried out to elucidate the underlying interactions during the translocation of polypeptides through protein channels. For this we used OmpF from the outer cell membrane of E. coli and arginine-based peptides of different charges, lengths and covalently linked polyethylene glycol as a model system. In order to reveal the fast kinetics of peptide binding, we performed a temperature scan. Together with the voltage-dependent single-channel conductance, we quantify peptide binding and translocation.
Subject(s)
Models, Molecular , Peptides/metabolism , Porins/metabolism , Cell Membrane/metabolism , Electrophysiological Phenomena , Escherichia coli/cytology , Kinetics , Peptides/chemistry , Polyethylene Glycols/chemistry , Porins/chemistry , Protein Conformation , Protein Transport , Temperature , ThermodynamicsABSTRACT
The TOM protein complex facilitates the transfer of nearly all mitochondrial preproteins across outer mitochondrial membranes. Here we characterized the effect of temperature on facilitated translocation of a mitochondrial presequence peptide pF1ß. Ion current fluctuations analysis through single TOM channels revealed thermodynamic and kinetic parameters of substrate binding and allowed determining the energy profile of peptide translocation. The activation energy for the on-rate and off-rate of the presequence peptide into the TOM complex was symmetric with respect to the electric field and estimated to be about 15 and 22 kT per peptide. These values are above that expected for free diffusion of ions in water (6 kT) and reflect the stronger interaction in the channel. Both values are in the range for typical enzyme kinetics and suggest one process without involving large conformational changes within the channel protein.
