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PEDS Advance Access originally published online on October 6, 2004
Protein Engineering Design and Selection 2004 17(9):665-672; doi:10.1093/protein/gzh078
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Protein Engineering, Design & Selection vol. 17 no. 9 © Oxford University Press 2004; all rights reserved

Deletions of single extracellular loops affect pH sensitivity, but not voltage dependence, of the Escherichia coli porin OmpF

Arnaud Baslé, Randa Qutub, Mahsa Mehrazin, Jamie Wibbenmeyer and Anne H. Delcour1

Department of Biology and Biochemistry, University of Houston, 369 Science and Research Building 2, Houston, TX 77205-5001, USA

1 To whom correspondence should be addressed. E-mail: adelcour{at}uh.edu

The molecular basis for the voltage and pH dependence of the Escherichia coli OmpF porin activity remains unknown. The L3 loop was previously shown not be involved in voltage dependence. Here we used seven OmpF mutants where single extracellular loops, except L3, were deleted one at a time. The proteins are expressed at levels comparable to wild-type and purified as trimers. Wild-type and mutant proteins were inserted into planar lipid bilayers for electrophysiological measurement of their activity. Current–voltage relationships show the typical porin channel closure at voltages greater than the critical voltage. Measurements of critical voltages for the seven deletion mutants showed no significant differences relative to wild-type, hence eliminating the role of single loops in voltage sensitivity. However, deletions of loops L1, L7 or L8 affected the tendency of channels to close at acidic pH. Wild-type channels close more readily at acidic pH and their open probability is decreased by ~60% at pH 4.0 relative to pH 7.0. For mutants lacking loop L1, L7 or L8, the channel open probability was found not to be significantly different at pH 4.0 than at pH 7.0. The other deletion mutants retained a pH sensitivity similar to the wild-type channel. Possible mechanistic scenarios for the voltage- and pH dependence of E.coli OmpF porin are discussed based on these results.

Received June 11, 2004; revised August 25, 2004; accepted September 28, 2004.

Edited by Hagan Bayley


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