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Seminars
Fibrillation Kinetics of Recombinant Human Insulin at Interfaces and with Osmolytes: Experiments and Kinetic Modeling
Professor Georges Belfort
Department of Chemical and Biological Engineering
Rensselaer Polytechnic Institute
When: Thursday, April 19, 2007
Time: 2:30 PM to 3:30 PM
Where: 1070 Duncall Hall
Abstract:
Amyloid fibrillation is the process of native soluble proteins misfolding into insoluble fibrils comprising of cross-β-sheets and has received wide attention due to its substantial physiological relevance and the complexity of the underlying physical and chemical reactions. At present, more than 20 amyloidogenic diseases including Alzheimer’s disease, Parkinson’s disease, and prion–associated encephalopathies have been found to share fibril formation as the common cause. Human insulin is chosen as a model molecule for our study because (i) it is associated with a clinical syndrome, injection-localized amyloidosis, (ii) it is a member of the class of fibril forming proteins that loses its zinc- coordinated hexameric structure to form monomers that then fibrils, (iii) of its well-characterized in vitro fibrillation kinetics under well-defined solution conditions (2 mg/ml, pH 1.6 and 65ºC), and (iv) fibril formation is a problem in commercial isolation and purification of insulin at low pH values of 1-3.
Here, we investigate the influence of suspended solid interfaces and dissolved osmolytes (sugars) on the kinetics of insulin fibrillation. For apolar solid substrates, insulin nucleation is speeded up while the growth rate of fibers is unaffected. However, in the presence of sugars, the fibrillation process (both the lag-time and the rate constant to form fibrils) is delayed and appears to correlate with the heats of solution at infinite dilution thereby supporting a preferential exclusion mechanism. With the recent focus on circular protofibrils and the suspicion that they may be toxic during amyloidosis, we have conducted a series of exploratory experiments during the lag phase when protofibrils are thought to be formed. This has included the use of small angle neutron scattering and sucrose gradient ultracentrifugation to investigate the kinetics of protofibrils formation. We also present a mathematical mechanistic model that simulates the phenomena by incorporating the physical chemistry of nucleation and growth dynamics. Estimated by nonlinear least square algorithms, we find rate constants that account for the ubiquitous sigmoidal responses of amyloidogenic proteins when they misfold. Finally, we address the thermodynamics of the nucleation process.
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