ABSTRACT
Chemical modification of ion channels has recently attracted attention due to their potential use in stochastic sensing and neurobiology. Among the available channel templates stable ß-barrel proteins have shown their potential for large scale chemical modifications due to their wide pore lumen. Ion-channel hybrids using the outer membrane protein OmpG were generated by S-alkylation with a synthetic modulator and functionally as well as structurally characterized. The dansyl moiety of the used modulator resulted in partial blockage of current though the OmpG channel with its gating characteristics mainly unaffected. The crystal structure of an OmpG-dansyl hybrid at 2.4Å resolution correlates this finding by showing that the modulator lines the inner walling of the OmpG pore. These results underline the suitability of OmpG as a structural base for the construction of stochastic sensors.
Subject(s)
Bacterial Outer Membrane Proteins/chemistry , Escherichia coli Proteins/chemistry , Porins/chemistry , Alkylation , Amino Acid Substitution , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Crystallography, X-Ray , Dansyl Compounds/chemistry , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Mutation , Porins/genetics , Porins/metabolism , Protein Structure, TertiaryABSTRACT
It's raining, it's porin: Fragment ligation of OmpF ion channels was achieved by using the split Psp-GBD Pol intein; this allowed reconstitution of active trimeric porin. In combination with cysteine modification at an internal position, the porin's conductance properties were altered.
Subject(s)
Escherichia coli , Porins/chemistry , Protein Splicing , Cysteine/metabolism , Electric Conductivity , Inteins , Models, Molecular , Porins/metabolism , Porosity , Protein Conformation , Protein RenaturationABSTRACT
The attachment of modulators to a trimeric porin ion channel was investigated (see picture of the trimer with a crown ether modulator (orange)). The interplay of modulator and protein is essential for the conformational heterogeneity of the hybrid channel. Single-site attachment in large pores is not sufficient to change the electrophysiological characteristics of the pores-such change requires additional noncovalent interactions or second-site attachments.
