Conductance and selectivity fluctuations in D127 mutants of the bacterial porin OmpF.
A recent molecular dynamics study questioned the protonation state and physiological role of aspartate 127 (D127) of E. coli porin OmpF. To address that question we isolated two OmpF mutants with D127 either neutralized (D127N) or replaced by a positively charged lysine (D127K). The charge state of the residue at position 127 has clear effects on both conductance and selectivity. The D127K but not the D127N mutant expresses resilient conductance and selectivity fluctuations. These fluctuations reflect, we think, either changes in the ionization state of K127 and/or transitions between unstable subconformations as induced by the electrostatic repulsion between two positively charged residues, K127 and the nearby R167. Our results slightly favor the view that in WT OmpF residue D127 is deprotonated. As for the role of D127 in OmpF functionality, the gating of both mutants shows very similar sensitivity toward voltage as WT OmpF. Moreover, the current fluctuations of the D127K mutant were observed also in the absence of an applied electric field. We therefore dismiss D127 as a key residue in the control mechanism of the voltage-dependent gating of OmpF.
Miedema
Henk
H
Biomade Technology Foundation, Nijenborgh 4, 9747 AG Groningen, The Netherlands. miedema@biomade.nl
Vrouenraets
Maarten
M
Wierenga
Jenny
J
Eisenberg
Bob
B
Schirmer
Tilman
T
Baslé
Arnaud
A
Meijberg
Wim
W
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry; D001703:Biophysics / Q000379:methods; D004594:Electrophysiology; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating; D007477:Ions; D008239:Lysine / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018272:Porins / Q000235:genetics* / Q000378:metabolism; D017433:Protein Structure, Secondary; D055672:Static Electricity
D001425:Bacterial Outer Membrane Proteins; D007477:Ions; D008565:Membrane Proteins; C109237:OmpF protein; D018272:Porins; D008239:Lysine