Use of a distant reporter group as evidence for a conformational change in a sensory receptor.

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Abstract
A highly sensitive method for demonstrating ligand-induced conformational changes in protein molecules in solution is described. The method utilizes an environmentally sensitive reporter group that is known to be distant from the active site. In the present application a conformational change is demonstrated in the galactose receptor of Salmonella typhimurium, involved in bacterial sensing and transport, by an extrinsic fluorophore, 5-iodoacetamidofluorescein, attached at a single methionine residue, and the intrinsic tryptophan fluorophore. Binding of the ligand galactose perturbs the microenvironment of the fluorescein and tryptophan, as shown by spectral and KI quenching changes. The distance between the 2 dyes is established by fluorescence energy transfer methods to be 41 .+-. 10 .ANG.. Since only 1 molecule of galactose binds/molecule of receptor and since the galactose molecule is only .apprx. 5 .ANG. in length, changes at 1 of these sites reflect the result of an indirect effect. Hence, there must be a ligand-induced conformational change that is propagated a minimum of 30 .ANG. through the receptor molecule.