The family of human diseases termed amyloidoses have the common feature that naturally occurring, normally innocuous soluble peptides or proteins assemble into particularly stable insoluble polymers which accumulate, increase in mass, and cause damage to surrounding tissue. Growth of the amyloid deposits is the hallmark pathological process of these diseases, but its mechanism is poorly understood. A major hurdle frustrating attempts to understand this process has been the lack of a simply and highly reproducible model system, which allows study of the deposition and dissociation kinetics at short timescale under a variety of conditions. Such a system would allow the essential thermodynamics of the processes to be worked out, and reaction steps vulnerable to therapeutic intervention identified. We have now developed such a system for studying the detailed kinetics of amyloid growth in a tissue-free system suitable for high throughput work. We have chosen to focus on the Ab amyloid of Alzheimer's disease, which makes up the brain senile plaques in this ailment, but a detailed knowledge of the deposition process will have implication for other diseases of protein misfolding. The determination of rate constants and activation entropies, enthalpies, and free energies of the template-mediated conformational changes from soluble to solid phase associated with amyloid growth, and the role of perturbations in structure and environment on these parameters, will be the major goal of these studies. Experimental determination of the kinetic parameters of a pathological folding transition involved in a human disease under physiological concentrations and conditions has not previously been possible. As a secondary aim, we will also examine the geometry of the peptide monomer within the amyloid fibril by photocrosslinking. Conclusions from the in vitro studies will be tested in human Alzheimer's disease brain sections.
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