DIRECT FORCE MEASUREMENTS OF INSULIN MONOMER-MONOMER INTERACTIONS

Direct measurement of the forces involved in protein-protein and prote in-receptor interactions can, in principle, provide insight necessary for the advancement of structural biology, molecular biology, and the development of therapeutic proteins. The protein insulin is illustrati ve in this respect as the mechanisms of insulin dimer dissociation and insulin-insulin receptor binding are crucial to the efficacy of insul in medications for the control of diabetes. Insulin molecules, modifie d with a photochemically active azido functionality on specific residu es, were attached to force microscope tips and opposing mica surfaces in configurations that would either favor or disfavor dimer formation. Force curve measurements performed in buffer solution revealed the co mplexity of the insulin monomer-monomer interaction with multiple unbi nding events occurring upon tip retraction, suggesting disruption of d iscrete molecular bonds at the monomer-monomer interface. Furthermore, the force curves exhibit long-range unbinding events, consistent with considerable elongation of the insulin molecule prior to dissociation . The unbinding forces observed in this study are the result of a comb ination of molecular disentanglement and dimer dissociation processes.