Protein Engineering, Vol 11, 797-802, Copyright © 1998 by Oxford University Press
N Liu and AH Delcour
Porins are trimeric channel-forming proteins of the outer membrane of
Escherichia coli. Each subunit contains 16 beta-strands forming a
transmembrane beta-barrel whose pore is constricted by the third
extracellular loop (L3). We investigated the effects of site-directed
mutations at two critical regions of the OmpC porin: (i) the D315A mutation
targets a key component of a putative hydrogen bond network linking the L3
loop to the adjacent barrel wall and (ii) the D118Q, R174Q and R92Q
mutations target putative salt bridges at the root of the L3 loop. We
purified the outer membrane fractions obtained from each mutant and
reconstituted them in liposomes suitable for electrophysiology. Patch clamp
experiments showed that the frequency of spontaneous transitions between
open and closed states is increased in the D315A, D118Q and R92Q mutants
but unchanged in the R174Q mutant. These transitions are not driven by
transmembrane voltage changes and represent the thermal oscillations
between functionally distinct conformations. The asymmetric
voltage-dependent inactivation of the channels is not affected by the
mutations, however, suggesting different molecular mechanisms for the
spontaneous and voltage- dependent gating processes. We propose that the
positioning or flexibility of the L3 loop across the pore, as governed by
the putative hydrogen-bond network and a salt bridge, play a role in
determining the frequency of spontaneous channel gating.
ARTICLES
The spontaneous gating activity of OmpC porin is affected by mutations of a putative hydrogen bond network or of a salt bridge between the L3 loop and the barrel
Department of Biology and Biochemistry, University of Houston, TX 77204- 5513, USA.
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