title: Chemical modification of matrix porin from Escherichia coli: probing the pore topology of a transmembrane protein.
abstract: Chemical modification of amino groups in matrix porin solubilized and purified from outer membranes of Escherichia coli in beta-octylglucoside was performed with eosin isothiocyanate and citraconic anhydride. At pH 7 8.5, the former reagent labeled a single amino group in the native protein, while more extensive derivatization was observed with increasing pH or upon denaturation. Citraconic anhydride modified approximately 12-14 residues in native porin and 15-16 of the total of 19 amino groups in the denatured state. Fluorescamine, another amine-specific reagent of intermediate size, derivatized 3 and 16 residues in the native and denatured states, respectively. These results indicate that reactive probes of various sizes may serve as indicators for the surface accessibility of reactive residues in matrix porin. The increased derivatization of lysyl residues at high pH (or in phosphate buffer) suggests the method's sensitivity to different conformational states of the protein. The extent of tyrosine modification (1-2 residues in the native, and approximately 22 in the denatured porin) depended on the state of protein folding, even with reagents of small size. The approach of using various probes with differing properties and specificities thus appears useful for the determination of membrane protein asymmetry, pore topology, and conformational states of transmembrane proteins.
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis*; D002949:Citraconic Anhydrides; D004801:Eosine Yellowish-(YS) / Q000031:analogs & derivatives; D004926:Escherichia coli / Q000032:analysis*; D005450:Fluorescamine; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000032:analysis*; D018272:Porins; D011487:Protein Conformation
chemical_list: D001426:Bacterial Proteins; D002949:Citraconic Anhydrides; D008565:Membrane Proteins; D018272:Porins; D005450:Fluorescamine; C012957:eosine-5-isothiocyanate; D004801:Eosine Yellowish-(YS)