Use of nonelectrolytes reveals the channel size and oligomeric constitution of the Borrelia burgdorferi P66 porin

• Published in: PloS one 2013-11, Vol.8 (11), p.e78272-e78272
• Main Authors: Bárcena-Uribarri, Iván, Thein, Marcus, Maier, Elke, Bonde, Mari, Bergström, Sven, Benz, Roland
• Format: Article
• Language: English
• Subjects: Analysis; Bacterial Proteins - chemistry; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Biology; black lipid bilayer; Blockage; Borrelia burgdorferi; Borrelia burgdorferi - chemistry; Cell Membrane - chemistry; Chemical reactions; Chlorides; Conductance; Constitution; E coli; Electric Conductivity; Electrophoresis, Polyacrylamide Gel; Engineering schools; Escherichia coli; Experiments; Gel electrophoresis; Gram-negative bacteria; Immunoblotting; Lipid bilayers; Lipid Bilayers - chemistry; Lipid Bilayers - metabolism; Lipids; Lyme disease; Maltose - chemistry; Medicine; Membrane conductance; Membrane Potentials - physiology; Membrane proteins; Membranes; Molecular biology; Noise; Nonelectrolytes; nutrient uptake; Oligosaccharides - chemistry; outer membrane transport; Polyethylene glycol; Polyethylene Glycols - chemistry; Pore formation; porin radius determination; Porins; Porins - chemistry; Porins - isolation & purification; Porins - metabolism; Potassium chloride; Potassium Chloride - chemistry; Protein Multimerization; Proteins; Resistance; Sodium lauryl sulfate; Spirochetes; Vector-borne diseases
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0078272

In the Lyme disease spirochete Borrelia burgdorferi, the outer membrane protein P66 is capable of pore formation with an atypical high single-channel conductance of 11 nS in 1 M KCl, which suggested that it could have a larger diameter than 'normal' Gram-negative bacterial porins. We studied the diameter of the P66 channel by analyzing its single-channel conductance in black lipid bilayers in the presence of different nonelectrolytes with known hydrodynamic radii. We calculated the filling of the channel with these nonelectrolytes and the results suggested that nonelectrolytes (NEs) with hydrodynamic radii of 0.34 nm or smaller pass through the pore, whereas neutral molecules with greater radii only partially filled the channel or were not able to enter it at all. The diameter of the entrance of the P66 channel was determined to be ≤1.9 nm and the channel has a central constriction of about 0.8 nm. The size of the channel appeared to be symmetrical as judged from one-sidedness of addition of NEs. Furthermore, the P66-induced membrane conductance could be blocked by 80-90% by the addition of the nonelectrolytes PEG 400, PEG 600 and maltohexaose to the aqueous phase in the low millimolar range. The analysis of the power density spectra of ion current through P66 after blockage with these NEs revealed no chemical reaction responsible for channel block. Interestingly, the blockage of the single-channel conductance of P66 by these NEs occurred in about eight subconductance states, indicating that the P66 channel could be an oligomer of about eight individual channels. The organization of P66 as a possible octamer was confirmed by Blue Native PAGE and immunoblot analysis, which both demonstrated that P66 forms a complex with a mass of approximately 460 kDa. Two dimension SDS PAGE revealed that P66 is the only polypeptide in the complex.