Involvement of a tryptophan residue in the binding site of escherichia coli galactose-binding protein

The galactose-binding protein, a component of the β-methyl galactoside transport system of Escherichia coli, undergoes a conformational change upon the binding of substrate. A variety of spectrophotometric techniques were employed in order to probe the nature of this change. All known substrates of the β-methyl galactoside transport system cause an alteration in the ultraviolet absorbance of the galactose-binding protein. The ultraviolet difference spectra produced by these substrates are similar but not identical and resemble solvent perturbation difference spectra of W-acetyltryptophan ethyl ester. In addition, solvent perturbation difference spectroscopy reveals that the exposure of external chromophores is not affected by binding of substrate. These results indicate that the substrate interacts directly with a tryptophan residue present in the binding site of the protein, and further, that the active site tryptophan is not accessible to the bulk solvent either in the presence or absence of substrate. However, substrate protects the protein from fluorescence quenching by KI. It is concluded that the alteration in surface charge of the protein which has been shown to accompany the binding of substrate causes this differential quenching of an external tryptophan. A mutant galactose-binding protein which does not exhibit an increase in electrical charge upon substrate binding is not protected from the quenching effects of KI by substrate, even though interaction of substrate with the active-site tryptophan still occurs. Thus, the substrate-dependent conformational change of the galactose-binding protein does not result in extensive refolding of the polypeptide chain, but rather is restricted to small alterations in the active site and an increase in the surface charge of the protein.