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. 2002 Mar;82(3):1667–1676. doi: 10.1016/S0006-3495(02)75517-3

Imaging the electrostatic potential of transmembrane channels: atomic probe microscopy of OmpF porin.

Ansgar Philippsen 1, Wonpil Im 1, Andreas Engel 1, Tilman Schirmer 1, Benoit Roux 1, Daniel J Müller 1
PMCID: PMC1301964  PMID: 11867478

Abstract

The atomic force microscope (AFM) was used to image native OmpF porin and to detect the electrostatic potential generated by the protein. To this end the OmpF porin trimers from Escherichia coli was reproducibly imaged at a lateral resolution of approximately 0.5 nm and a vertical resolution of approximately 0.1 nm at variable electrolyte concentrations of the buffer solution. At low electrolyte concentrations the charged AFM probe not only contoured structural details of the membrane protein surface but also interacted with local electrostatic potentials. Differences measured between topographs recorded at variable ionic strength allowed mapping of the electrostatic potential of OmpF porin. The potential map acquired by AFM showed qualitative agreement with continuum electrostatic calculations based on the atomic OmpF porin embedded in a lipid bilayer at the same electrolyte concentrations. Numerical simulations of the experimental conditions showed the measurements to be reproduced quantitatively when the AFM probe was included in the calculations. This method opens a novel avenue to determine the electrostatic potential of native protein surfaces at a lateral resolution better than 1 nm and a vertical resolution of approximately 0.1 nm.

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Selected References

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  1. Binnig G, Quate CF, Gerber C. Atomic force microscope. Phys Rev Lett. 1986 Mar 3;56(9):930–933. doi: 10.1103/PhysRevLett.56.930. [DOI] [PubMed] [Google Scholar]
  2. Butt H. J. Measuring electrostatic, van der Waals, and hydration forces in electrolyte solutions with an atomic force microscope. Biophys J. 1991 Dec;60(6):1438–1444. doi: 10.1016/S0006-3495(91)82180-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cowan S. W., Schirmer T., Rummel G., Steiert M., Ghosh R., Pauptit R. A., Jansonius J. N., Rosenbusch J. P. Crystal structures explain functional properties of two E. coli porins. Nature. 1992 Aug 27;358(6389):727–733. doi: 10.1038/358727a0. [DOI] [PubMed] [Google Scholar]
  4. Czajkowsky D. M., Iwamoto H., Cover T. L., Shao Z. The vacuolating toxin from Helicobacter pylori forms hexameric pores in lipid bilayers at low pH. Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):2001–2006. doi: 10.1073/pnas.96.5.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Drake B., Prater C. B., Weisenhorn A. L., Gould S. A., Albrecht T. R., Quate C. F., Cannell D. S., Hansma H. G., Hansma P. K. Imaging crystals, polymers, and processes in water with the atomic force microscope. Science. 1989 Mar 24;243(4898):1586–1589. doi: 10.1126/science.2928794. [DOI] [PubMed] [Google Scholar]
  6. Dutzler R., Rummel G., Albertí S., Hernández-Allés S., Phale P., Rosenbusch J., Benedí V., Schirmer T. Crystal structure and functional characterization of OmpK36, the osmoporin of Klebsiella pneumoniae. Structure. 1999 Apr 15;7(4):425–434. doi: 10.1016/s0969-2126(99)80055-0. [DOI] [PubMed] [Google Scholar]
  7. Engel A., Müller D. J. Observing single biomolecules at work with the atomic force microscope. Nat Struct Biol. 2000 Sep;7(9):715–718. doi: 10.1038/78929. [DOI] [PubMed] [Google Scholar]
  8. Engel A., Schoenenberger C. A., Müller D. J. High resolution imaging of native biological sample surfaces using scanning probe microscopy. Curr Opin Struct Biol. 1997 Apr;7(2):279–284. doi: 10.1016/s0959-440x(97)80037-1. [DOI] [PubMed] [Google Scholar]
  9. Heinz W. F., Hoh J. H. Relative surface charge density mapping with the atomic force microscope. Biophys J. 1999 Jan;76(1 Pt 1):528–538. doi: 10.1016/S0006-3495(99)77221-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hoenger A., Ghosh R., Schoenenberger C. A., Aebi U., Engel A. Direct in situ structural analysis of recombinant outer membrane porins expressed in an OmpA-deficient mutant Escherichia coli strain. J Struct Biol. 1993 Nov-Dec;111(3):212–221. doi: 10.1006/jsbi.1993.1051. [DOI] [PubMed] [Google Scholar]
  11. Honig B., Nicholls A. Classical electrostatics in biology and chemistry. Science. 1995 May 26;268(5214):1144–1149. doi: 10.1126/science.7761829. [DOI] [PubMed] [Google Scholar]
  12. Im W., Seefeld S., Roux B. A Grand Canonical Monte Carlo-Brownian dynamics algorithm for simulating ion channels. Biophys J. 2000 Aug;79(2):788–801. doi: 10.1016/S0006-3495(00)76336-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Karshikoff A., Spassov V., Cowan S. W., Ladenstein R., Schirmer T. Electrostatic properties of two porin channels from Escherichia coli. J Mol Biol. 1994 Jul 22;240(4):372–384. doi: 10.1006/jmbi.1994.1451. [DOI] [PubMed] [Google Scholar]
  14. Klebba P. E., Newton S. M. Mechanisms of solute transport through outer membrane porins: burning down the house. Curr Opin Microbiol. 1998 Apr;1(2):238–247. doi: 10.1016/s1369-5274(98)80017-9. [DOI] [PubMed] [Google Scholar]
  15. Lou K. L., Saint N., Prilipov A., Rummel G., Benson S. A., Rosenbusch J. P., Schirmer T. Structural and functional characterization of OmpF porin mutants selected for larger pore size. I. Crystallographic analysis. J Biol Chem. 1996 Aug 23;271(34):20669–20675. [PubMed] [Google Scholar]
  16. McLaughlin S. The electrostatic properties of membranes. Annu Rev Biophys Biophys Chem. 1989;18:113–136. doi: 10.1146/annurev.bb.18.060189.000553. [DOI] [PubMed] [Google Scholar]
  17. Müller D. J., Amrein M., Engel A. Adsorption of biological molecules to a solid support for scanning probe microscopy. J Struct Biol. 1997 Jul;119(2):172–188. doi: 10.1006/jsbi.1997.3875. [DOI] [PubMed] [Google Scholar]
  18. Müller D. J., Engel A. The height of biomolecules measured with the atomic force microscope depends on electrostatic interactions. Biophys J. 1997 Sep;73(3):1633–1644. doi: 10.1016/S0006-3495(97)78195-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Müller D. J., Fotiadis D., Engel A. Mapping flexible protein domains at subnanometer resolution with the atomic force microscope. FEBS Lett. 1998 Jun 23;430(1-2):105–111. doi: 10.1016/s0014-5793(98)00623-1. [DOI] [PubMed] [Google Scholar]
  20. Müller D. J., Fotiadis D., Scheuring S., Müller S. A., Engel A. Electrostatically balanced subnanometer imaging of biological specimens by atomic force microscope. Biophys J. 1999 Feb;76(2):1101–1111. doi: 10.1016/S0006-3495(99)77275-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nikaido H., Saier M. H., Jr Transport proteins in bacteria: common themes in their design. Science. 1992 Nov 6;258(5084):936–942. doi: 10.1126/science.1279804. [DOI] [PubMed] [Google Scholar]
  22. Roux B., Bernèche S., Im W. Ion channels, permeation, and electrostatics: insight into the function of KcsA. Biochemistry. 2000 Nov 7;39(44):13295–13306. doi: 10.1021/bi001567v. [DOI] [PubMed] [Google Scholar]
  23. Roux B., MacKinnon R. The cavity and pore helices in the KcsA K+ channel: electrostatic stabilization of monovalent cations. Science. 1999 Jul 2;285(5424):100–102. doi: 10.1126/science.285.5424.100. [DOI] [PubMed] [Google Scholar]
  24. Roux B., Simonson T. Implicit solvent models. Biophys Chem. 1999 Apr 5;78(1-2):1–20. doi: 10.1016/s0301-4622(98)00226-9. [DOI] [PubMed] [Google Scholar]
  25. Saint N., Lou K. L., Widmer C., Luckey M., Schirmer T., Rosenbusch J. P. Structural and functional characterization of OmpF porin mutants selected for larger pore size. II. Functional characterization. J Biol Chem. 1996 Aug 23;271(34):20676–20680. [PubMed] [Google Scholar]
  26. Saint N., Prilipov A., Hardmeyer A., Lou K. L., Schirmer T., Rosenbusch J. P. Replacement of the sole histidinyl residue in OmpF porin from E. coli by threonine (H21T) does not affect channel structure and function. Biochem Biophys Res Commun. 1996 Jun 5;223(1):118–122. doi: 10.1006/bbrc.1996.0855. [DOI] [PubMed] [Google Scholar]
  27. Sanner M. F., Olson A. J., Spehner J. C. Reduced surface: an efficient way to compute molecular surfaces. Biopolymers. 1996 Mar;38(3):305–320. doi: 10.1002/(SICI)1097-0282(199603)38:3%3C305::AID-BIP4%3E3.0.CO;2-Y. [DOI] [PubMed] [Google Scholar]
  28. Schabert F. A., Engel A. Reproducible acquisition of Escherichia coli porin surface topographs by atomic force microscopy. Biophys J. 1994 Dec;67(6):2394–2403. doi: 10.1016/S0006-3495(94)80726-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schabert F. A., Henn C., Engel A. Native Escherichia coli OmpF porin surfaces probed by atomic force microscopy. Science. 1995 Apr 7;268(5207):92–94. doi: 10.1126/science.7701347. [DOI] [PubMed] [Google Scholar]
  30. Schirmer T. General and specific porins from bacterial outer membranes. J Struct Biol. 1998;121(2):101–109. doi: 10.1006/jsbi.1997.3946. [DOI] [PubMed] [Google Scholar]
  31. Schirmer T., Phale P. S. Brownian dynamics simulation of ion flow through porin channels. J Mol Biol. 1999 Dec 17;294(5):1159–1167. doi: 10.1006/jmbi.1999.3326. [DOI] [PubMed] [Google Scholar]
  32. Sharp K. A., Honig B. Electrostatic interactions in macromolecules: theory and applications. Annu Rev Biophys Biophys Chem. 1990;19:301–332. doi: 10.1146/annurev.bb.19.060190.001505. [DOI] [PubMed] [Google Scholar]
  33. Weiss M. S., Abele U., Weckesser J., Welte W., Schiltz E., Schulz G. E. Molecular architecture and electrostatic properties of a bacterial porin. Science. 1991 Dec 13;254(5038):1627–1630. doi: 10.1126/science.1721242. [DOI] [PubMed] [Google Scholar]

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