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Disulfide bond tethering of extracellular loops does not affect the closure of OmpF porin at acidic pH

Lookup NU author(s): Dr Arnaud Basle

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Abstract

The permeability of the outer membrane of gram-negative bacteria is essentially controlled by pore-forming proteins of the porin family. The trimeric E. coli porin OmpF is assembled as a triple β-barrel, where each monomer contains a central pore and extracellular loops. Electrophysiological analysis of the behavior of OmpF at acidic pH reveals that the protein undergoes a conformational change leading to the sequential step-wise closure of the three monomers. A previous atomic force microscopy study suggested that the conformational change might be due to a bending of extracellular loops over the pore opening, and loop deletion experiments suggested that loops L1, L7, and L8 are involved. In order to test the hypothesis for loop movement, we engineered a series of double cysteine mutants in loops L1, L6, L7 and L8 in order to create disulfide bonds linking two loops to each other, or the two branches of a loop, or a loop to the β-barrel. Five out of the six mutants showed the formation of the disulfide bond. However, none of these had an altered response to acidic pH relative to the wildtype channel. Although we cannot dismiss the possibility that the mobility restriction introduced by each disulfide bond was too localized to impact a more global conformational change of the three loops, the fact that all of the different types of disulfide bond tethering were similarly ineffective suggests that the extracellular loops L1, L7, and L8 may not undergo a major acidicpH induced conformational change leading to channel closure. © 2010 Wiley-Liss, Inc.


Publication metadata

Author(s): Wager B, Baslé A, Delcour A

Publication type: Article

Publication status: Published

Journal: Proteins: Structure, Function and Bioformatics

Year: 2010

Volume: 78

Issue: 14

Pages: 2886-2894

Print publication date: 29/06/2010

ISSN (print): 0887-3585

ISSN (electronic): 1097-0134

Publisher: John Wiley & Sons Ltd

URL: http://dx.doi.org/10.1002/prot.22807

DOI: 10.1002/prot.22807


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