Osmolyte controlled nucleation of insulin amyloid fibrillation

BIOT 485

Arpan Nayak, nayaka@rpi.edu, Howard Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, 110 8th street, Troy, NY 12180, Chuang-Chung Lee, Department of Chemical Engineering, Massachussets Institute of Technology, Cambridge, MA 02139, Gregory J. McRae, mcrae@mit.edu, Department of Chemical Engineering, Massachusetts Institute of Technology, Room 66-372, 77 Massachusetts Avenue, Cambridge, MA 02139-4307, and Georges Belfort, Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12180-3590.
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 a common phenomenon. 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 oligomers and 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 dissolved osmolytes on the kinetics of insulin fibrillation. While sugars (stabilizing osmolytes) delay the onset of fibrillation, urea and guanidine (destabilizing osmolytes) accelerate it. The inhibiting effect of sugars is correlated with their heats of solution and is explained by the theory of preferential exclusion of sugar molecules from protein surfaces. We show that osmolytes with higher neutral surface area have a better protecting effect on the native protein. A mathematical mechanistic model that simulates the phenomena by incorporating the chemical reactions of nucleation and growth dynamics is presented. Using model fits of the experimental data, the rate constants and Gibbs free energy for nucleation are estimated. Both increased or decreased in presence of destabilizing and stabilizing osmolytes, respectively. Taken all together, these results provide a thermodynamic basis for the mechanism of amyloid fibrillation and provide insight into the role of sugar-based excipients for pharmaceutical formulations.
 

Biophysical and Biomolecular Symposium: Protein Aggregation
2:00 PM-5:25 PM, Thursday, August 23, 2007 BCEC -- 107C, Oral

Division of Biochemical Technology

The 234th ACS National Meeting, Boston, MA, August 19-23, 2007