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Bordetella pertussis major outer membrane porin protein forms small, anion-selective channels in lipid bilayer
Abstract:
The major outer membrane protein of molecular weight 40,000 (the 40K protein) of a virulent isolate of Bordetella pertussis was purified to apparent homogeneity. The purified protein formed an oligomer band (of apparent molecular weight 90,000) on sodium dodecyl sulfate-polyacrylamide gels after solubilization at low temperatures. The porin function of this protein was characterized by the black lipid bilayer method. The 40K protein formed channels smaller than all other constitutive major outer membrane porins studied to date. The average single-channel conductance in 1 M KCl was 0.56 nS. This was less than a third of the conductance previously observed for Escherichia coli porins. Zero-current potential measurements made of the porin to determine its ion selectivity revealed the porin to be more than 100-fold selective for anions over cations. The single-channel conductance was measured as a function of salt concentration. The data could be fitted to a Lineweaver-Burk plot suggesting an anion binding site with a Kd of 1.17 M Cl- and a maximum possible conductance through the channel of 1.28 nS.
Insights
The 40K protein from Bordetella pertussis acts as a small anion-selective channel, differing significantly from other bacterial porins. This research details its unique biophysical properties and ion transport characteristics.
Area of Science:
- Microbiology
- Biophysics
- Structural Biology
Background:
- Bordetella pertussis is a significant human pathogen.
- Outer membrane proteins, including porins, play crucial roles in bacterial physiology and interaction with the environment.
- Understanding the structure-function relationship of bacterial porins is vital for developing novel antimicrobial strategies.
Purpose of the Study:
- To purify and characterize the major 40,000 molecular weight (40K) outer membrane protein from Bordetella pertussis.
- To investigate the porin function and ion transport properties of the purified 40K protein.
- To compare the channel characteristics of the 40K protein with other known bacterial porins.
Main Methods:
- Purification of the 40K protein to homogeneity.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to assess protein aggregation.
- Black lipid bilayer method to characterize porin function, including single-channel conductance and ion selectivity.
- Zero-current potential measurements to determine ion selectivity.
- Salt concentration-dependent conductance measurements fitted to a Lineweaver-Burk plot.
Main Results:
- The 40K protein was purified and shown to form oligomers.
- The 40K protein functions as a porin, forming channels smaller than other characterized bacterial porins.
- The channel exhibits high anion selectivity (>100-fold over cations).
- Single-channel conductance in 1 M KCl was 0.56 nS, significantly lower than E. coli porins.
- Analysis revealed an anion binding site with a Kd of 1.17 M Cl- and a maximum conductance of 1.28 nS.
Conclusions:
- The 40K protein of Bordetella pertussis is a unique, small, anion-selective porin.
- Its distinct biophysical properties suggest a specialized role in nutrient uptake or molecular transport.
- Further studies are warranted to elucidate its precise physiological function and potential as a drug target.