TY - JOUR
T1 - Ca2+ selectivity of a chemically modified OmpF with reduced pore volume
AU - Miedema, Henk
AU - Vrouenraets, Maarten
AU - Wierenga, Jenny
AU - Gillespie, Dirk
AU - Eisenberg, Bob
AU - Meijberg, Wim
AU - Nonner, Wolfgang
PY - 2006/12
Y1 - 2006/12
N2 - 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+.
AB - 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+.
UR - http://www.scopus.com/inward/record.url?scp=33845393202&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33845393202&partnerID=8YFLogxK
U2 - 10.1529/biophysj.106.087114
DO - 10.1529/biophysj.106.087114
M3 - Article
C2 - 16997866
AN - SCOPUS:33845393202
VL - 91
SP - 4392
EP - 4400
JO - Biophysical Journal
JF - Biophysical Journal
SN - 0006-3495
IS - 12
ER -