Use of nonelectrolytes reveals the channel size and oligomeric constitution of the Borrelia burgdorferi P66 porin.
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.
School of Engineering and Science, Jacobs University Bremen, Bremen, Germany ; Rudolf-Virchow-Center, DFG-Research Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany.
Iván
I
Bárcena-Uribarri
Marcus
M
Thein
Elke
E
Maier
Mari
M
Bonde
Sven
S
Bergström
Roland
R
Benz
D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D025065:Borrelia burgdorferi / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry*; D004553:Electric Conductivity; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D008320:Maltose / Q000737:chemistry; D008564:Membrane Potentials / Q000502:physiology; D009844:Oligosaccharides / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D018272:Porins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011189:Potassium Chloride / Q000737:chemistry; D055503:Protein Multimerization
D001426:Bacterial Proteins; D008051:Lipid Bilayers; D009844:Oligosaccharides; C107893:Oms66 protein, Borrelia; D018272:Porins; C016549:maltohexaose; D011092:Polyethylene Glycols; D011189:Potassium Chloride; D008320:Maltose