AFFINITY-CHROMATOGRAPHIC PURIFICATION OF 16 CYSTEINE-SUBSTITUTED MALTOPORIN VARIANTS - THIOL REACTIVITY AND CROSS-LINKING IN AN OUTER-MEMBRANE PROTEIN OF ESCHERICHIA-COLI

被引:15
作者
FRANCIS, G [1 ]
BRENNAN, L [1 ]
FERENCI, T [1 ]
机构
[1] UNIV SYDNEY,DEPT MICROBIOL,SYDNEY,NSW 2006,AUSTRALIA
基金
澳大利亚研究理事会;
关键词
THIOL REACTIVITY; CROSS-LINKING; MALTOPRIN VARIANT; CYSTEINE SUBSTITUTION; MEMBRANE PROTEIN; MEMBRANE TOPOLOGY; AFFINITY CHROMATOGRAPHY; (ESCHERICHIA-COLI);
D O I
10.1016/0005-2736(91)90029-8
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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[C-14]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 > 265 > 30 approximately equal-to 103 approximately equal-to 120 approximately equal-to 154 approximately equal-to 382 > 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.
引用
收藏
页码:89 / 96
页数:8
相关论文
共 29 条
[1]   TRANSMEMBRANE PROTEIN-STRUCTURE - SPIN LABELING OF BACTERIORHODOPSIN MUTANTS [J].
ALTENBACH, C ;
MARTI, T ;
KHORANA, HG ;
HUBBELL, WL .
SCIENCE, 1990, 248 (4959) :1088-1092
[2]   PORE FORMATION BY LAMB OF ESCHERICHIA-COLI IN LIPID BILAYER-MEMBRANES [J].
BENZ, R ;
SCHMID, A ;
NAKAE, T ;
VOSSCHERPERKEUTER, GH .
JOURNAL OF BACTERIOLOGY, 1986, 165 (03) :978-986
[3]   LINKER MUTAGENESIS IN THE GENE OF AN OUTER-MEMBRANE PROTEIN OF ESCHERICHIA-COLI, LAMB [J].
BOUGESBOCQUET, B ;
VILLARROYA, H ;
HOFNUNG, M .
JOURNAL OF CELLULAR BIOCHEMISTRY, 1984, 24 (03) :217-228
[4]   PROBING THE TOPOLOGY OF A BACTERIAL-MEMBRANE PROTEIN BY GENETIC INSERTION OF A FOREIGN EPITOPE - EXPRESSION AT THE CELL-SURFACE [J].
CHARBIT, A ;
BOULAIN, JC ;
RYTER, A ;
HOFNUNG, M .
EMBO JOURNAL, 1986, 5 (11) :3029-3037
[5]   FURTHER SEQUENCE-ANALYSIS OF THE PHAGE-LAMBDA RECEPTOR-SITE - POSSIBLE IMPLICATIONS FOR THE ORGANIZATION OF THE LAMB PROTEIN IN ESCHERICHIA-COLI-K12 [J].
CHARBIT, A ;
CLEMENT, JM ;
HOFNUNG, M .
JOURNAL OF MOLECULAR BIOLOGY, 1984, 175 (03) :395-401
[6]   MALTOSE TRANSPORT AND STARCH BINDING IN PHAGE-RESISTANT POINT MUTANTS OF MALTOPORIN - FUNCTIONAL AND TOPOLOGICAL IMPLICATIONS [J].
CHARBIT, A ;
GEHRING, K ;
NIKAIDO, H ;
FERENCI, T ;
HOFNUNG, M .
JOURNAL OF MOLECULAR BIOLOGY, 1988, 201 (03) :487-496
[7]  
Creighton T. E., 1989, PROTEIN STRUCTURE PR, P155
[8]   MUTATIONS AFFECTING ANTIGENIC DETERMINANTS OF AN OUTER-MEMBRANE PROTEIN OF ESCHERICHIA-COLI [J].
DESAYMARD, C ;
DEBARBOUILLE, M ;
JOLIT, M ;
SCHWARTZ, M .
EMBO JOURNAL, 1986, 5 (06) :1383-1388
[9]   TISSUE SULFHYDRYL GROUPS [J].
ELLMAN, GL .
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 1959, 82 (01) :70-77
[10]   GLOBAL FLEXIBILITY IN A SENSORY RECEPTOR - A SITE-DIRECTED CROSS-LINKING APPROACH [J].
FALKE, JJ ;
KOSHLAND, DE .
SCIENCE, 1987, 237 (4822) :1596-1600