Ca2+ selectivity of a chemically modified OmpF with reduced pore volume.
We studied an E. coli OmpF mutant (LECE) containing both an EEEE-like locus, typical of Ca(2+) channels, and an accessible and reactive cysteine. After chemical modification with the cysteine-specific, negatively charged (-1e) reagents MTSES or glutathione, this LECE mutant was tested for Ca(2+) versus alkali metal selectivity. Selectivity was measured by conductance and zero-current potential. Conductance measurements showed that glutathione-modified LECE had reduced conductance at Ca(2+) mole fractions <10(-3). MTSES-modified LECE did not. Apparently, the LECE protein is (somehow) a better Ca(2+) chelator after modification with the larger glutathione. Zero-current potential measurements revealed a Ca(2+) versus monovalent cation selectivity that was highest in the presence of Li(+) and lowest in the presence of Cs(+). Our data clearly show that after the binding of Ca(2+) the LECE pore (even with the bulky glutathione present) is spacious enough to allow monovalent cations to pass. Theoretical computations based on density functional theory combined with Poisson-Nernst-Planck theory and a reduced pore model suggest a functional separation of ionic pathways in the pore, one that is specific for small and highly charged ions, and one that accepts preferentially large ions, such as Cs(+).
Miedema
Henk
H
Biomade Technology Foundation, Nijenborgh, Groningen, The Netherlands.
Vrouenraets
Maarten
M
Wierenga
Jenny
J
Gillespie
Dirk
D
Eisenberg
Bob
B
Meijberg
Wim
W
Nonner
Wolfgang
W
D002118:Calcium / Q000737:chemistry*; D002414:Cations, Monovalent / Q000737:chemistry; D002586:Cesium / Q000737:chemistry; D003198:Computer Simulation; D003545:Cysteine / Q000737:chemistry; D004553:Electric Conductivity; D004560:Electricity; D005978:Glutathione / Q000737:chemistry; D015640:Ion Channel Gating; D008051:Lipid Bilayers / Q000737:chemistry; D008094:Lithium / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D018272:Porins / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D012964:Sodium / Q000737:chemistry
D002414:Cations, Monovalent; D008051:Lipid Bilayers; C109237:OmpF protein; D018272:Porins; D002586:Cesium; D008094:Lithium; D012964:Sodium; D005978:Glutathione; D003545:Cysteine; D002118:Calcium