Affinity-chromatographic purification of sixteen cysteine-substituted maltoporin variants: thiol reactivity and cross-linking in an outer membrane protein of Escherichia coli.
Wild-type and 16 variant maltoporins with site-directed cysteine substitutions at 14 sites were purified by a novel one-step affinity-chromatographic procedure. The trimer stability of purified proteins with C22S, C38S and G103C substitutions was reduced compared to wild-type maltoporin. Quantitative labelling with N-ethyl[14C]maleimide, cross-linking with bifunctional bismaleimides and disulphide formation was used to test the reactivity of cysteines in the folded protein. The maleimide reactivity of the residues was in the order: 152 approximately equal to 153 greater than 265 greater than 30 approximately equal to 103 approximately equal to 120 approximately equal to 154 approximately equal to 382 greater than 57 approximately equal to 146, with the other sites (22, 38, 97, 184) poorly labelled. Only cysteines at 152 or 153 permitted the formation of inter-subunit disulphide bonds suggesting these residues are located within 0.5-0.9 nm of each other in homotrimers of maltoporin. S152C and S153C as well as S154C permitted the formation of inter-subunit cross-links using bifunctional bismaleimides. The cross-linkability and the high reactivity to N-ethylmaleimide of the 150 region was consistent with the current model of the structure of maltoporin in the outer membrane; the reactivity of the other sites is also discussed within the context of this model.
Francis
G
G
Department of Microbiology, University of Sydney, N.S.W., Australia.
Brennan
L
L
Ferenci
T
T
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis*; D002846:Chromatography, Affinity; D003545:Cysteine; D004926:Escherichia coli / Q000032:analysis*; D005033:Ethylmaleimide / Q000494:pharmacology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010084:Oxidation-Reduction; D018272:Porins; D011991:Receptors, Virus / Q000235:genetics / Q000302:isolation & purification*; D013438:Sulfhydryl Compounds / Q000032:analysis*; D013696:Temperature
D001425:Bacterial Outer Membrane Proteins; D018272:Porins; D011991:Receptors, Virus; D013438:Sulfhydryl Compounds; C016532:maltoporins; D003545:Cysteine; D005033:Ethylmaleimide