ESCHERICHIA-COLI K12 ARABINOSE-BINDING PROTEIN MUTANTS WITH ALTERED TRANSPORT-PROPERTIES

被引:8
作者
KEHRES, DG [1 ]
HOGG, RW [1 ]
机构
[1] CASE WESTERN RESERVE UNIV,SCH MED,DEPT MOLEC BIOL & MICROBIOL,CLEVELAND,OH 44106
关键词
ESCHERICHIA-COLI; PERIPLASMIC TRANSPORT; PROTEIN PROTEIN INTERACTIONS; SITE-DIRECTED MUTAGENESIS;
D O I
10.1002/pro.5560011213
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
The arabinose-binding protein (ABP) of Escherichia coli binds L-arabinose in the periplasm and delivers it to a cytoplasmic membrane complex consisting of the AraG and AraH proteins, for uptake into the cell. To study the interaction between the soluble and membrane components of this periplasmic transport system, regions of the ABP surface containing the opening of the arabinose-binding cleft were subjected to site-directed mutagenesis. Thirty-eight ABP variants containing one to three amino acid substitutions were recovered. ABP variants were expressed with wild-type AraG and AraH from a plasmid, in a strain lacking the chromosomal araFGH operon, and the whole cell uptake parameters, V(en) (maximum initial velocity of arabinose entry) and K(en) (concentration of arabinose yielding half-maximal entry) were determined. Twenty-four mutants had normal V(en) values, 3 mutants had V(en) and K(en) values twice wild type, and 11 mutants had V(en) and K(en) values 20-50% of wild type. Binding proteins that had altered uptake properties were each expressed, processed, and localized to the periplasm at levels equivalent to wild type. The mutant binding proteins behaved the same as wild type during purification, and each had a K(d) (dissociation constant for bound arabinose) comparable to that of wild-type ABP. Mutations that resulted in altered uptake identified nine amino acids surrounding the arabinose-binding cleft, all of which are charged in the wild-type protein, and all of whose side chains project outward from the cleft. The evidence suggests that this surface of the binding protein and these nine charged loci play a major role in ABP interactions with the membrane complex.
引用
收藏
页码:1652 / 1660
页数:9
相关论文
共 31 条
[1]   SIMPLE, RAPID, AND QUANTITATIVE RELEASE OF PERIPLASMIC PROTEINS BY CHLOROFORM [J].
AMES, GF ;
PRODY, C ;
KUSTU, S .
JOURNAL OF BACTERIOLOGY, 1984, 160 (03) :1181-1183
[2]   ENERGY COUPLING IN BACTERIAL PERIPLASMIC PERMEASES [J].
AMES, GFL ;
JOSHI, AK .
JOURNAL OF BACTERIOLOGY, 1990, 172 (08) :4133-4137
[3]   FUNCTIONAL MAPPING OF THE SURFACE OF ESCHERICHIA-COLI RIBOSE-BINDING PROTEIN - MUTATIONS THAT AFFECT CHEMOTAXIS AND TRANSPORT [J].
BINNIE, RA ;
ZHANG, H ;
MOWBRAY, S ;
HERMODSON, MA .
PROTEIN SCIENCE, 1992, 1 (12) :1642-1651
[4]   ONE-STEP PREPARATION OF COMPETENT ESCHERICHIA-COLI - TRANSFORMATION AND STORAGE OF BACTERIAL-CELLS IN THE SAME SOLUTION [J].
CHUNG, CT ;
NIEMELA, SL ;
MILLER, RH .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1989, 86 (07) :2172-2175
[5]   DIRECT LINEAR PLOT - NEW GRAPHICAL PROCEDURE FOR ESTIMATING ENZYME KINETIC-PARAMETERS [J].
EISENTHAL, R ;
CORNISH-BOWDEN, A .
BIOCHEMICAL JOURNAL, 1974, 139 (03) :715-720
[6]   STRUCTURE OF THE L-ARABINOSE-BINDING PROTEIN FROM ESCHERICHIA-COLI AT 2.4-A RESOLUTION [J].
GILLILAND, GL ;
QUIOCHO, FA .
JOURNAL OF MOLECULAR BIOLOGY, 1981, 146 (03) :341-362
[7]  
HANAHAN D, 1983, J MOL BIOL, V166, P577
[8]  
HOGG RW, 1982, METHOD ENZYMOL, V90, P463
[9]   L-ARABINOSE TRANSPORT AND L-ARABINOSE BINDING-PROTEIN OF ESCHERICHIA-COLI [J].
HOGG, RW .
JOURNAL OF SUPRAMOLECULAR STRUCTURE, 1977, 6 (03) :411-417
[10]   HIGH-AFFINITY L-ARABINOSE TRANSPORT OPERON - GENE-PRODUCT EXPRESSION AND MESSENGER-RNAS [J].
HORAZDOVSKY, BF ;
HOGG, RW .
JOURNAL OF MOLECULAR BIOLOGY, 1987, 197 (01) :27-35