| During the protein folding process, nascent polypeptide chains usually proceed through the correct folding pathway to achieve the native conformation either spontaneously or with the help of co-factors, ions and molecular chaperones. Disruption of one or more of the stabilizing interactions that facilitate proper folding can lead to misfolding or aggregation of protein molecules. The significance of protein folding has generated enormous interest, in the pursuit both to understand the factors associated with the protein folding pathway and to design new therapies for misfolding diseases such as cystic fibrosis and Alzheimer's. It also has tremendous importance in biotechnology and pharmaceutical industries engaged in recombinant protein production.Detergents are commonly used to refold unfolded proteins because they sequester the exposed hydrophobic residues that lead to aggregation. However, dissolution of the protein-detergent complex is difficult and leads to poor refolding yields. Since the length of the hydrocarbon chain is likely related to the strength of the protein-detergent complex, an alternative approach to solve the aggregation problem is to synthesize partially fluorous surfactants that contain tunable short hydrocarbon and non-interacting fluorocarbon components. It is our hypothesis that partially fluorinated surfactants will have sufficient affinity for hydrophobic residues exposed in an unfolded state to prevent protein aggregation, yet still be readily displaced by dialysis, thus providing high yields of renatured protein.In this dissertation, a series of hemifluorinated surfactants containing varying degrees of hydrophobic and fluorophilic character has been synthesized and characterized. This synthetic approach will allow the affinity of hemifluorinated surfactant for the protein to be "fine tuned." In an attempt to understand the physical properties of these surfactants in solution, the critical micelle concentration (CMC) and hydrodynamic radii (Rh) were determined. Results indicate that hemifluorinated surfactants have lower CMC and R h values than their hydrocarbon analogues. To examine the efficacy of hemifluorinated surfactants as renaturing additives, bovine carbonic anhydrase (CAB) was selected because this protein is prone to aggregation during the folding process. Protein renaturation studies show that anionic hemifluorinated surfactants are effective as renaturing additives for CAB below their CMC, providing high refolding yields for thermally and chemically denatured protein. The results indicate that these surfactants provide improved refolding yields at low as well as high concentrations of CAB compared to their hydrocarbon analogs. Furthermore, this approach has been applied to renature matrix metalloproteinase-13 (MMP-13) inclusion bodies overproduced in host cells, which is the most important problem associated with recombinant expression systems. A systematic study of the interactions between these surfactants and various proteins or inclusion bodies will provide a more rational approach to protein renaturation.During our studies with hemifluorinated surfactants, we observed their potential spermicidal applications. Many spermicidal products contain detergents such as nonoxynol-9 (N-9). Although N-9 effectively kills sperm, it causes damage to surrounding tissues that lead to specific health risks for females. An alternative approach to detergents could be hemifluorinated surfactants. Initial in vitro studies were performed on sperm and cervical/HeLa cells in the presence of hemifluorinated surfactants. Preliminary data indicates that these hemifluorinated surfactants could potentially aid in the development of new strategies for contraception. Overall, the work contained herein has delineated a novel approach to tackle the protein aggregation problem, and an alternative to traditional spermicides that will have less deleterious impact on normal tissues. |
