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Macromolecular assemblies: Human gamma-crystallin protein, glutamic acid bottle brushes, and hyaluronic acid gels
Macromolecular assemblies constitute the world in which we live. The work contained within this thesis has studied three different types of macromolecular assemblies: human gamma-crystallin protein aggregation, the synthesis of glutamic acid bottle brushes, and cross-linked hyaluronic acid hydrogels. The in vitro study of human lens gamma-crystallin protein aggregation is the main component of this thesis. Separate projects that aid the body of this work include a description for the synthesis of glutamic acid bottle brush macromolecules and novel cross-linked networks of hyaluronic acid hydrogels. Cataract is the number one cause of blindness worldwide. Despite being a widespread disease, the epidemiology of cataract is still largely unknown. Cataracts are protein aggregates consisting of alpha-, beta-, and gamma-crystallin protein which are the major components of the lens in the human eye. In this work, in vitro investigations of protein-protein interactions were performed on dilute solutions of recombinant human alpha- and gamma-crystallin protein using dynamic light scattering technique. It was discovered that in phosphate buffered solutions, gamma-crystallin exists as two distinct populations of unaggregated and large aggregated protein. On the other hand, alpha-crystallin protein does not aggregate, but forms small oligomeric assemblies. Upon mixing alpha- and gamma-crystallin the aggregation of gamma-crystallin was removed. The impact of temperature, protein concentration, the reducing agent dithiothreitol, salt concentration, and pH on gamma-crystallin were all subsequently investigated. It was concluded that in vitro aggregation of gamma-crystallin protein arises from non-covalent electrostatic interactions. To further investigate the electrostatic hypothesis, point mutations were performed on human gamma-crystallin in an attempt to prevent protein aggregation. Using recombinant DNA technology, twelve different gammaS-crystallin protein mutants were created. The mutations were designed to change the proteins overall surface charge by substituting positively charged amino acids with neutral hydrophilic amino acids. The aggregation behavior of the mutant proteins was then studied by dynamic light scattering. It was observed that all gamma-crystallin mutants resulted in continued aggregation. The result suggests that the in vitro aggregation behavior of gamma-crystallin is likely due to electrostatic interactions between specific amino acids not probed in this work. Due to the time consuming nature of point mutations, chemical modifications of gamma-crystallin were subsequently performed in an alternative method to disrupt electrostatic interactions which are believed to cause gamma-crysatllin protein aggregation. GammaD- and GammaS-crystallin proteins were chemically modified with seven different molecules. Chemical modifications were characterized with a combination of mass spectroscopy, circular dichroism spectroscopy, and dynamic light scattering. The results demonstrated that modifying positively charged amino acids of human gamma-crystallin protein with poly(ethylene) glycol prevented in vitro protein aggregation. The chemical modification of lens crystallin protein provides a possible route by which protein aggregation and ultimately cataract can be prevented, arrested or reversed. In a separate project, glutamic acid bottle brushes were synthesized for future translocation experiments. Translocation is viewed as a potential method by which genomic DNA can be sequenced. In an attempt to understand the effect of polymer diameter on translocation kinetics, bottle brush polymers with varying thicknesses were synthesized. The complex synthesis and subsequent characterization by nuclear magnetic resonance, atomic force microscopy, capillary electrophoresis, static and dynamic light scattering are described herein. In summary, glutamic acid bottle brushes of three different thicknesses were successfully synthesized, characterized, and purified. Finally, the novel synthesis of thiol cross-linked hyaluronic acid hydrogels is described. Hyaluronic acid is a polysaccharide which is of interest for potential biomedical applications. The biopolymer was modified with cystamine and placed under reducing conditions which provides free thiol groups capable of crosslinking under oxidizing conditions. The synthesis, characterization, and gelation procedure for the cross-linked hyaluronic acid hydrogels is described in detail. The modified hyaluronic acid is a material capable of in situ gelation.^
Mohr, Benjamin Georg Robert, "Macromolecular assemblies: Human gamma-crystallin protein, glutamic acid bottle brushes, and hyaluronic acid gels" (2013). Doctoral Dissertations Available from Proquest. AAI3603124.