69.    Kinetic Analysis of AB Fibril Elongation1

Analytical Biochemistry (2004) In press

We used surface plasmon resonance biosensors to evaluate the kinetics associated with the initial events of Ab fibril elongation. Fibrils were immobilized on the sensor chip surface and extended by exposure to soluble Ab(1-40) peptide. The fibril surfaces bound Congo red, a marker for B-sheet structures, and exhibited a slow linear background decay that is consistent with fibril depolymerization. Sonicated fibrils supported elongation better than unsonicated fibrils, which is consistent with fibril extension reactions. The kinetic data revealed that peptide association and dissociation occurred in multiple steps. Kinetic rate constants for fibril extension were determined by globally fitting the response data with a three-step polymerization model. In the first step, the soluble peptide binds to the growing fibril tip in a readily reversible reaction. The subsequent steps likely allow bound peptide to be stabilized into the growing fiber through post-binding transitional events. Using a mutant peptide, F19P AB(1-40), we illustrate how the biosensor assay can be used to probe structure/function relationships of fibril elongation.