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Electrostatic effects in aggregation of crystallin proteins

Deniz Elizabeth Civay, University of Massachusetts Amherst

Abstract

The three projects utilized polymer physics theories to investigate polymer aggregation mechanics. Dynamic light scattering (DLS), static light scattering (SLS) and small angle light scattering (SALS) were the primary characterization tools. The goal of the first project was to study the aggregation of bovine βL-crystallin and apply that knowledge towards cataract formation, which is caused by aggregation of the crystallins. The first series of experiments characterized the kinetics of α-crystallin and β L-crystallin in water at room temperature. α-crystallin’s equilibrium hydrodynamic radius value was kinetically independent. β L-crystallin formed an aggregate with an Rh that was kinetically dependent. The packing structure of the aggregate formed by βL-crystallin was determined to be loosely packed using SLS. α -crystallin was uniquely demonstrated to be a chaperone in a way that indicated electrostatics played a significant role in aggregation. The role of electrostatics led to an investigation into sodium chloride. Sodium chloride proved to reduce the βL-crystallin aggregate size. The next series of experiments simulated biological conditions using a phosphate buffered saline (PBS). The experiments were performed at 35°C. α -crystallin and βL-crystallin were shown to be kinetically independent and demonstrate equilibrium Rh values on the time scale that the experiments were performed. A pH study revealed that multiple size-scales were present only at physiological pH. Above and below physiological pH, only two aggregate size-scales existed. A charge model was made of β L-crystallin to compare theory with experimental results. The future goal of project is to reproduce these experiments with human crystallins. In the second project, by changing the order and arrangement of β-spiral elastin (E) and α -helical COMPcc (C) the macroscopic structure was controlled. The EC diblock exhibited a fast and slow mode below the transition temperature of 25°C and single mode behavior above the transition. Phase separation occurred above the transition. CE showed three different size-scales below the transition of 15°C and demonstrated spinodal decomposition above the transition. The ECE triblock demonstrated bimodal behavior below the transition of 25°C and one micellar size above the transition. α-helical COMPcc has the ability to bind to small molecules, making the findings from this project instrumental in creating a drug delivery vehicle. The third project investigated sodium polystyrene sulfonate and polyethylene oxidepolypropylene oxide-polyethylene oxide in solution. Both systems self-assemble into aggregate structures at specific conditions. The significant difference between these two polymers is that sodium polystyrene sulfonate is a polyelectrolyte. It is well known that aggregate structures can be formed by variation in temperature and concentration. However, by having a charged polymer in solution with a neutral polymer the aggregate structure can also be controlled by changing the pH and adding salt to the solution, as was performed in the first project. The third project is an excellent conclusion to the previous two because it allows for the aggregate structure to be controlled even more so than in the previous projects by mediating the polydispersity index, molecular weight and concentration of each component. Each project focused on a different method of mediating the aggregate structure. A better understanding of aggregation has applications in industry and medicine. Polymer physics theory is instrumental in understanding aggregation mechanics.

Subject Area

Polymer chemistry|Biophysics|Plastics

Recommended Citation

Civay, Deniz Elizabeth, "Electrostatic effects in aggregation of crystallin proteins" (2011). Doctoral Dissertations Available from Proquest. AAI3482599.
https://scholarworks.umass.edu/dissertations/AAI3482599

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