Analyzing the Self-Organizing Mechanism of Lysozyme Amyloid Fiber Formation

Abstract

The formation of amyloid fibers involves a number of different intermediates. By using separation techniques and analysis methods such as dielectric spectroscopy, AFM, and TEM, the aggregation steps of fiber formation were analyzed. An Agilent 4294A impedance analyzer and an Agilent 16452A liquid test fixture over a frequency range of 40Hz to 30MHz was used for dielectric spectroscopy. We approach amyloid fiber formation using the newly introduced colloidal model [1]. This model suggests that proteins aggregate into uniformly sized nano-spheres, driven by surface energy minimization. The uniform spheres then behave like a mono-dispersed colloidal suspension. Once the spheres have reached their critical diameter it is observed from microscopy that the colloidal growth stops. At this point the attractive forces that favor agglomeration are balanced by the barrier potential forces that retard agglomeration. The fully developed nucleation units then assemble in a linear fashion before finally evolving into mature amyloid fibers. The model postulates that the linear assembly arises from dipole-dipole interaction between nano-spheres. We analyze this assembly process in vitro using lysozyme from chicken egg whites in an acidic environment. In vivo, lysozyme has a propensity to form amyloid fibers in systemic amyloidosis diseases. Lysozyme amyloid fibers are synthesized in vitro and separated into samples according to particle size. Our separation techniques yielded three samples: 1. a solution with a high concentration of monomeric lysozyme and small oligomers, 2. a solution composed of colloidal spheres and short fibers, and 3. a solution with a high concentration of mature amyloid fibers. The existence of these species in the three samples was confirmed with AFM, TEM, and Thioflavin-T binding assays. Results of dielectric analysis indicate intermediate sized aggregates have a higher dipole moment than small aggregates. [1] S. Xu, Amyloid, 14, 119 (2007).