Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1998 Jul;7(7):1603–1611. doi: 10.1002/pro.5560070714

Porin mutants with new channel properties.

B Schmid 1, L Maveyraud 1, M Krömer 1, G E Schulz 1
PMCID: PMC2144057  PMID: 9684893

Abstract

The general diffusion porin from Rhodopseudomonas blastica was produced in large amounts in Escherichia coli inclusion bodies and (re)natured to the exact native structure. Here, we report on 13 mutants at the pore eyelet giving rise to new diffusion properties as measured in planar lipid bilayer experiments. The crystal structures of seven of these mutants were established. The effects of charge-modifying mutations at the pore eyelet are consistent with the known selectivity for cations. Deletions of 16 and 27 residues of the constriction loop L3 resulted in labile trimers and pores. The reduction of the eyelet cross section by introducing tryptophans gave rise to a closely correlated decrease of the conductivities. A mutant with six newly introduced tryptophans in the eyelet closed its pore in a defined manner within seconds under a voltage of 20 mV, suggesting the existence of two states. The results indicate that the pore can be engineered in a rational manner.

Full Text

The Full Text of this article is available as a PDF (3.3 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bauer K., Struyvé M., Bosch D., Benz R., Tommassen J. One single lysine residue is responsible for the special interaction between polyphosphate and the outer membrane porin PhoE of Escherichia coli. J Biol Chem. 1989 Oct 5;264(28):16393–16398. [PubMed] [Google Scholar]
  2. Benson S. A., Occi J. L., Sampson B. A. Mutations that alter the pore function of the OmpF porin of Escherichia coli K12. J Mol Biol. 1988 Oct 20;203(4):961–970. doi: 10.1016/0022-2836(88)90121-0. [DOI] [PubMed] [Google Scholar]
  3. Bishop N. D., Lea E. J., Mobasheri H., Spiro S. Altered voltage sensitivity of mutant OmpC porin channels. FEBS Lett. 1996 Feb 5;379(3):295–298. doi: 10.1016/0014-5793(95)01535-3. [DOI] [PubMed] [Google Scholar]
  4. Braha O., Walker B., Cheley S., Kasianowicz J. J., Song L., Gouaux J. E., Bayley H. Designed protein pores as components for biosensors. Chem Biol. 1997 Jul;4(7):497–505. doi: 10.1016/s1074-5521(97)90321-5. [DOI] [PubMed] [Google Scholar]
  5. Gill S. C., von Hippel P. H. Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem. 1989 Nov 1;182(2):319–326. doi: 10.1016/0003-2697(89)90602-7. [DOI] [PubMed] [Google Scholar]
  6. Gokce I., Bainbridge G., Lakey J. H. Stabilising and destabilising modifications of cysteines in the E. coli outer membrane porin protein OmpC. FEBS Lett. 1997 Jul 14;411(2-3):201–205. doi: 10.1016/s0014-5793(97)00690-x. [DOI] [PubMed] [Google Scholar]
  7. Jeanteur D., Schirmer T., Fourel D., Simonet V., Rummel G., Widmer C., Rosenbusch J. P., Pattus F., Pagès J. M. Structural and functional alterations of a colicin-resistant mutant of OmpF porin from Escherichia coli. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10675–10679. doi: 10.1073/pnas.91.22.10675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kreusch A., Neubüser A., Schiltz E., Weckesser J., Schulz G. E. Structure of the membrane channel porin from Rhodopseudomonas blastica at 2.0 A resolution. Protein Sci. 1994 Jan;3(1):58–63. doi: 10.1002/pro.5560030108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kreusch A., Schulz G. E. Refined structure of the porin from Rhodopseudomonas blastica. Comparison with the porin from Rhodobacter capsulatus. J Mol Biol. 1994 Nov 11;243(5):891–905. doi: 10.1006/jmbi.1994.1690. [DOI] [PubMed] [Google Scholar]
  10. Lamzin V. S., Wilson K. S. Automated refinement of protein models. Acta Crystallogr D Biol Crystallogr. 1993 Jan 1;49(Pt 1):129–147. doi: 10.1107/S0907444992008886. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Misra R., Benson S. A. Isolation and characterization of OmpC porin mutants with altered pore properties. J Bacteriol. 1988 Feb;170(2):528–533. doi: 10.1128/jb.170.2.528-533.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Murshudov G. N., Vagin A. A., Dodson E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr. 1997 May 1;53(Pt 3):240–255. doi: 10.1107/S0907444996012255. [DOI] [PubMed] [Google Scholar]
  14. Nicholls A., Sharp K. A., Honig B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins. 1991;11(4):281–296. doi: 10.1002/prot.340110407. [DOI] [PubMed] [Google Scholar]
  15. Nikaido H. Porins and specific diffusion channels in bacterial outer membranes. J Biol Chem. 1994 Feb 11;269(6):3905–3908. [PubMed] [Google Scholar]
  16. Ponder J. W., Richards F. M. Tertiary templates for proteins. Use of packing criteria in the enumeration of allowed sequences for different structural classes. J Mol Biol. 1987 Feb 20;193(4):775–791. doi: 10.1016/0022-2836(87)90358-5. [DOI] [PubMed] [Google Scholar]
  17. Przybylski M., Glocker M. O., Nestel U., Schnaible V., Blüggel M., Diederichs K., Weckesser J., Schad M., Schmid A., Welte W. X-ray crystallographic and mass spectrometric structure determination and functional characterization of succinylated porin from Rhodobacter capsulatus: implications for ion selectivity and single-channel conductance. Protein Sci. 1996 Aug;5(8):1477–1489. doi: 10.1002/pro.5560050804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rocque W. J., McGroarty E. J. Structure and function of an OmpC deletion mutant porin from Escherichia coli K-12. Biochemistry. 1990 Jun 5;29(22):5344–5351. doi: 10.1021/bi00474a020. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Schmid B., Krömer M., Schulz G. E. Expression of porin from Rhodopseudomonas blastica in Escherichia coli inclusion bodies and folding into exact native structure. FEBS Lett. 1996 Feb 26;381(1-2):111–114. doi: 10.1016/0014-5793(96)00080-4. [DOI] [PubMed] [Google Scholar]
  22. Schulz G. E. Bacterial porins: structure and function. Curr Opin Cell Biol. 1993 Aug;5(4):701–707. doi: 10.1016/0955-0674(93)90143-e. [DOI] [PubMed] [Google Scholar]
  23. Schulz G. E. Porins: general to specific, native to engineered passive pores. Curr Opin Struct Biol. 1996 Aug;6(4):485–490. doi: 10.1016/s0959-440x(96)80113-8. [DOI] [PubMed] [Google Scholar]
  24. Soares C. M., Björkstén J., Tapia O. L3 loop-mediated mechanisms of pore closing in porin: a molecular dynamics perturbation approach. Protein Eng. 1995 Jan;8(1):5–12. doi: 10.1093/protein/8.1.5. [DOI] [PubMed] [Google Scholar]
  25. Srikumar R., Dahan D., Arhin F. F., Tawa P., Diederichs K., Coulton J. W. Porins of Haemophilus influenzae type b mutated in loop 3 and in loop 4. J Biol Chem. 1997 May 23;272(21):13614–13621. doi: 10.1074/jbc.272.21.13614. [DOI] [PubMed] [Google Scholar]
  26. Taylor J. W., Ott J., Eckstein F. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8765–8785. doi: 10.1093/nar/13.24.8765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Van Gelder P., Saint N., van Boxtel R., Rosenbusch J. P., Tommassen J. Pore functioning of outer membrane protein PhoE of Escherichia coli: mutagenesis of the constriction loop L3. Protein Eng. 1997 Jun;10(6):699–706. doi: 10.1093/protein/10.6.699. [DOI] [PubMed] [Google Scholar]
  28. Weiss M. S., Schulz G. E. Structure of porin refined at 1.8 A resolution. J Mol Biol. 1992 Sep 20;227(2):493–509. doi: 10.1016/0022-2836(92)90903-w. [DOI] [PubMed] [Google Scholar]
  29. Weiss M. S., Wacker T., Weckesser J., Welte W., Schulz G. E. The three-dimensional structure of porin from Rhodobacter capsulatus at 3 A resolution. FEBS Lett. 1990 Jul 16;267(2):268–272. doi: 10.1016/0014-5793(90)80942-c. [DOI] [PubMed] [Google Scholar]
  30. de Cock H., van Blokland S., Tommassen J. In vitro insertion and assembly of outer membrane protein PhoE of Escherichia coli K-12 into the outer membrane. Role of Triton X-100. J Biol Chem. 1996 May 31;271(22):12885–12890. doi: 10.1074/jbc.271.22.12885. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES