Ca2+ selectivity of a chemically modified OmpF with reduced pore volume

Henk Miedema, Maarten Vrouenraets, Jenny Wierenga, Dirk Gillespie, Bob Eisenberg, Wim Meijberg, Wolfgang Nonner

Research output: Contribution to journalArticlepeer-review

46 Scopus citations

Abstract

We studied an E. coli OmpF mutant (LECE) containing both an EEEE-like locus, typical of Ca2+ 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 Ca2+ versus alkali metal selectivity. Selectivity was measured by conductance and zero-current potential. Conductance measurements showed that glutathione-modified LECE had reduced conductance at Ca2+ mole fractions <10-3. MTSES-modified LECE did not. Apparently, the LECE protein is (somehow) a better Ca2+ 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 Ca2+ 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+.

Original languageEnglish (US)
Pages (from-to)4392-4400
Number of pages9
JournalBiophysical journal
Volume91
Issue number12
DOIs
StatePublished - Dec 2006

ASJC Scopus subject areas

  • Biophysics

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