Transport properties of the galactose-binding protein of Escherichia coli. Substrate-induced conformational change.

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 ( 10eg to 3 .lO’ M) yields an apparent Z‘&i,s of lo-’ M and at higher galactose concentrations (3 .lO-’ to lo-” M) an apparent Kdiss of 10e5 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 10e4 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 1OV M glucose and lop4 M (D-glyceryl)-1-/I?-D-galactopyranoside substrates of the transport system. Methyl-1-0-/3-D-galactopyranoside, methyl-lthio-fl-D-galactopyranoside, and isopropyl-1-thio-o-D-galactopyranoside at concentrations of lop4 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-/3-D-galactopyranoside occurs at half-maximal total sugar concentrations of lOW, lo-“, and lo-” 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 fl conformation.