Transport Properties of the Galactose-binding Protein of Escherichia coli: SUBSTRATE-INDUCED CONFORMATIONAL CHANGE

Abstract

Abstract The galactose-binding protein, a necessary component of a bacterical transport system, is shown by three independent methods to undergo a conformational change upon binding of substrate. 1. A Lineweaver-Burk plot of the binding data obtained by equilibrium dialysis at a protein concentration of 0.4 mg per ml shows heterogenous behavior. Extrapolation of the data at low galactose concentrations (10-8 to 3·10-7 m) yields an apparent Kdiss of 10-7 m and at higher galactose concentrations (3·10-7 to 10-5 m) an apparent Kdiss of 10-5 m. A Scatchard plot of the binding data extrapolated to high galactose concentrations indicates two binding sites per 36,000 molecular weight. 2. The presence of 10-4 m galactose during analytical polyacrylamide gel electrophoresis at pH 8.4 results in a changed electrophoretic mobility of the binding protein. This change is also observed in the presence of 10-4 m glucose and 10-4 m (d-glyceryl)-1-β-d-galactopyranoside substrates of the transport system. Methyl-1-O-β-d-galactopyranoside, methyl-1-thio-β-d-galactopyranoside, and isopropyl-1-thio-β-d-galactopyranoside at concentrations of 10-4 m do not cause the altered electrophoretic mobility. 3. The fluorescence of the protein is increased up to 13.5% in the presence of substrate between 310 to 350 nm when excited at 290 nm. The increase of fluorescence observed with galactose, glucose, and (d-glyceryl)-1-β-d-galactopyranoside occurs at half-maximal total sugar concentrations of 10-6, 10-6, and 10-5 m, respectively. The substrate-induced conformational change does not result in a change of the molecular weight of the protein as measured by high speed equilibrium centrifugation and sieve chromatography through Bio-Gel P-150. The conformational change of the protein cannot be observed by optical rotatory dispersion, circular dichroism, and infrared spectroscopy. These latter spectroscopic methods show, however, that the galactose-binding protein exists to a large extent in β conformation. Purified binding protein from a transport-negative mutant does not undergo the substrate-induced conformational changes measured by binding, electrophoretic mobility, and fluorescence.