The ionization state of D37 in E. coli porin OmpF and the nature of conductance fluctuations in D37 mutants.
Understanding the flow of ions through E. coli porin outer membrane protein F (OmpF) requires knowledge of the charge state of all titratable residues located along the permeation pathway. Earlier theoretical studies proved successful in the calculation of the pK values of most residues. The (apparent) pK of Asp37 (D37), on the other hand, appeared rather sensitive to the (unknown) protein dielectric used. We addressed the protonation state of D37 experimentally by replacing D37 with a (neutral) valine. This D37V mutant expressed reduced cation selectivity, in agreement with the view that D37 in wild-type (WT) OmpF is fully ionized, i.e., deprotonated. The introduction of a (positively charged) arginine at position 37 evoked current fluctuations. Similar behavior was observed in the D37K mutant and the cysteine mutants D37C-MTSEA and D37C-MTSET. Nontitratable [2-(trimethylammonium)ethyl]-methanethiosulfonate (MTSET) carries a permanent and pH-independent charge of 1e, implying that the fluctuations of the D37C-MTSET mutant do not represent (de)protonation reactions of MTSET. We therefore conclude that these fluctuations reflect transitions between conformational substates evoked by structural instabilities due to the positive charge at that particular position in the pore lumen. Based on the similarities between D37C-MTSET fluctuations and those seen in the other mutants, notably D37K, the underlying mechanism of these fluctuations may be (essentially) the same in all four mutants studied.
Biomade Technology Foundation, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
Maarten
M
Vrouenraets
Henk
H
Miedema
D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry / Q000235:genetics; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics*; D002412:Cations / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004553:Electric Conductivity; D004594:Electrophysiology; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D005020:Ethyl Methanesulfonate / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating; D009154:Mutation; D018272:Porins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D013997:Time Factors
D002412:Cations; C109237:OmpF protein; D018272:Porins; D001224:Aspartic Acid; D001120:Arginine; D005020:Ethyl Methanesulfonate; D003545:Cysteine