Structurally Defective Galactose-binding Protein Isolated from a Mutant Negative in the β-Methylgalactoside Transport System of Escherichia coli

Abstract

Abstract The galactose-binding protein was isolated and purified from a mutant, EH3039, defective in the β-methylgalactoside transport system. The purification consisted of cold osmotic shock, ammonium sulfate precipitation, electrofocusing, Sephadex G-100 chromatography, preparative polyacrylamide gel electrophoresis, and chromatography through Bio-Gel P-150. The mutant protein cross-reacts with antibodies against wild type galactose-binding protein and exhibits identical molecular weight as measured by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Peptide mapping of the tryptic digests shows that the mutant binding protein has changed pattern in comparison with the wild type protein, demonstrating a change in the polypeptide chain of the protein upon mutation of the wild type. The protein possesses binding affinity for d-galactose with a dissociation constant higher than 0.1 mm. The conformational change observed by polyacrylamide gel electrophoresis with the wild type binding protein upon binding of d-galactose does not occur with the mutant protein. However, it does show an increase in fluorescence at 336 nm (excitation 290 nm) in the presence of high concentrations of d-galactose, as does the wild type. The half-maximal increase of fluorescence occurs at 3.4 mm d-galactose, in contrast to the wild type value of 1 µm. d-Glucose and d-glyceryl-1-β-d-galactoside, which are active with wild type binding protein, also give rise to fluorescence increase in the mutant protein at concentrations higher than 1 mm. The mutant strain, EH3039, has been reverted to a transport-positive phenotype. The galactose-binding protein of this revertant was also purified and shown to exhibit properties characteristic for the wild type protein: binding towards galactose has a Kdiss of 0.2 µm; binding of substrate induces a conformational change resulting in an increase of electrophoretic mobility of the protein at pH 8.3; binding of substrate also increases the tryptophan fluorescence of the protein at a half-maximal substrate concentration of 1 µm. The simultaneous alteration in the structure and activity of the galactose-binding protein upon mutation of a wild type strain (W3092cy-) to a transport-negative mutant (EH3039) and its reversion to a transport-positive strain (LA39) clearly demonstrates the essential role of the galactose-binding protein in the function of the β-methylgalactoside transport system.