ABSTRACT
The role of the outer-membrane channel from a mycolic acid containing Gram-positive bacteria Nocardia farcinica, which forms a hydrophilic pathway across the cell wall, was characterized. Single channel electrophysiology measurements and liposome swelling assays revealed the permeation of hydrophilic solutes including sugars, amino acids and antibiotics. The cation selective N. farcinica channel exhibited strong interaction with the positively charged antibiotics; amikacin and kanamycin, and surprisingly also with the negatively charged ertapenem. Voltage dependent kinetics of amikacin and kanamycin interactions were studied to distinguish binding from translocation. Moreover, the importance of charged residues inside the channel was investigated using mutational studies that revealed rate limiting interactions during the permeation.
Subject(s)
Anti-Bacterial Agents/chemistry , Bacterial Proteins/chemistry , Cell Membrane/chemistry , Liposomes/chemistry , Nocardia/chemistry , Porins/chemistry , Amikacin/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biological Transport , Cell Membrane/metabolism , Cell Wall/chemistry , Ertapenem , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Hydrophobic and Hydrophilic Interactions , Kanamycin/chemistry , Models, Molecular , Mutagenesis, Site-Directed , Mutation , Mycolic Acids/chemistry , Nocardia/metabolism , Porins/genetics , Porins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Static Electricity , Structural Homology, Protein , beta-Lactams/chemistryABSTRACT
We investigated translocation of cationic peptides through nanochannels derived from the Gram-positive bacterium Nocardia farcinica at the single-molecule level. The two subunits NfpA and NfpB form a hetero-oligomeric cation selective channel. On the basis of amino acid comparison we performed homology modeling and obtained a channel structurally related to MspA of Mycobacterium smegmatis. The quantitative single-molecule measurements provide an insight into transport processes of solutes through nanochannels. High-resolution ion conductance measurements in the presence of peptides of different charge and length revealed the kinetics of peptide binding. The observed asymmetry in peptide binding kinetics indicated a unidirectional channel insertion in the lipid bilayer. In the case of cationic peptides, the external voltage acts as a driving force that promotes the interaction of the peptide with the channel surface. At low voltage, the peptide just binds to the channel, whereas at higher voltage, the force is strong enough to pull the peptide across the channel. This allows distinguishing quantitatively between peptide binding and translocation through the channel.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Nanostructures , Nocardia , Peptides/metabolism , Protein Multimerization , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Models, Molecular , Porins/chemistry , Porins/metabolism , Protein Structure, Quaternary , Protein Transport , Sequence HomologyABSTRACT
We characterize the rate-limiting interaction of the antibiotic enrofloxacin with OmpF, a channel from the outer cell wall of Escherichia coli . Reconstitution of a single OmpF trimer into planar lipid membranes allows measurement of the ion current through the channel. Penetration of antibiotics causes ion current blockages, and their frequency allows a conclusion on the kinetics of channel entry and exit. In contrast to other antibiotics, enrofloxacin is able to block the OmpF channel for several milliseconds, reflecting high affinities comparable to substrate-specific channels such as the maltodextrin-specific maltoporin. Surprisingly, the presence of a divalent ion such as Mg(2+) leads to fast flickering with an increase in the rates of association and dissociation. All-atom computer modeling provides the most probable pathway able to identify the relevant rate-limiting interaction during antibiotic permeation. Mg(2+) has a high affinity for the aspartic acid at the 113 position (D113) in the center of the OmpF intracellular binding site. Therefore, the presence of Mg(2+) reverses the charge and enrofloxacin may cross the constriction region in its favorable orientation with the carboxylic group first.
Subject(s)
Anti-Bacterial Agents/chemistry , Magnesium/pharmacology , Porins/chemistry , Anti-Bacterial Agents/metabolism , Aspartic Acid , Binding Sites/drug effects , Models, Molecular , Permeability , Porins/metabolism , Protein Structure, TertiaryABSTRACT
The permeation of water soluble molecules across cell membranes is controlled by channel-forming proteins and, in particular, the channel surface determines the selectivity. An adequate method to study the properties of these channels is electrophysiology and, in particular, analyzing the ion current fluctuation in the presence of permeating solutes. Ion current fluctuation analysis provides information on possible interactions of solutes with the channel surface. Due to the limited time resolution, fast permeation events are not visible using standard techniques. Here, we demonstrate that miniaturization of the lipid bilayer; varying the temperature or changing the solvent may enhance the resolution. Although electrophysiology is considered as a single molecule technique, it does not provide atomic resolution. Molecular details of solute permeation can be revealed by combining electrophysiology and all-atom computer modeling; these methods include ion conductance, selectivity, ion pair formation, and rate limiting interactions of the solute with the channel walls during permeation.
