Competitive labeling as an approach to defining the binding surfaces of proteins: binding of monomeric insulin to lipid bilayers.

Abstract

The free monomeric form of insulin is known to adsorb strongly to many different surfaces. A question of physiological relevance for which no previous studies have been reported is whether the monomeric form of insulin binds to lipid bilayers. In order to answer this question, it is necessary to carry out studies at the very dilute concentrations (less than 10(-6) M) necessary to obtain this species. We have approached this problem by applying the method of competitive labeling [Hefford, M.A., Evans, R.M., Oda, G., & Kaplan, H. (1985) Biochemistry 24, 867-874] to study insulin at concentrations as low as 3 X 10(-8) M, in the presence and absence of large unilamellar liposomes. With 1-fluoro-2,4-dinitrobenzene as the labeling reagent, the relative chemical reactivities of the functional groups of insulin were found to decrease markedly when insulin was incubated with liposomes consisting of egg lecithin and cholesterol (2:1 mol/mol) in 1.0 M KCl, pH 7.5 at 37 degrees C. The decrease for each functional group was found to directly correlate with its proximity to the dimer-forming surface of the monomer. It is concluded that insulin binds to lipid bilayers in a specific orientation, with the dimer-forming surface interacting with the bilayer. These results demonstrate the feasibility of applying competitive labeling to obtain structure-function relationships of membrane-interactive proteins in general and monomeric insulin in particular.