Date of Award
Doctor of Philosophy (PhD)
Understanding the fundamental science of aggregation and surface adsorption of proteins is critical for developing protein-based drugs, understanding aggregation-induced diseases and for preventing biofouling that impacts a wide range of industries. In spite of the advances in sensitive and high-throughput analytical methods, the commercialization of protein drugs has been impeded by poor solubility, inevitable aggregation, and difficulty in purification. The primary aim of this dissertation is to modify proteins covalently to induce folding in random coil sequences, prevent aggregation, surface adsorption and for increasing the thermal stability against aggregation of proteins.
The first chapter describes the chemistry for modifying the lysine residues in proteins with alditols (organic kosmotropes), via an amide bond using the traditional N-hydroxysuccinimide (NHS) activated carboxylic acids or via an efficient water-driven ligation generating a squaramide linkage. Conjugating such organic kosmotropes to proteins via a squaramide linkage, results in the formation of a hydrolytically stable bond together with higher amount of modification when compared to amide bond formation using N-activated carboxylic acids.
The second chapter presents a general approach for preventing both protein aggregation and surface adsorption by modifying proteins with beta-cyclodextrins (betaCD) via a squaramide linkage. As compared to native unmodified proteins, the beta-cyclodextrin-modified proteins (lysozyme and RNase A) exhibited significant reduction in aggregation, surface adsorption and increase in thermal stability against aggregation.
The third chapter describes the covalent modification of a random coil mutant (L44A) of the coiled coil domain of cartilage oligomeric matrix protein (COMPcc) with squarate derivatives. The covalent modification with betaCD-amino squarates or methyl squarate alone folded the random coil into an a-helix. The COMPcc is capable of binding small molecule such ascurcumin, retinol and vitamin D. Curcumin exhibited an increase in the fluorescence signal upon binding to L44A(betaCD)n as compared to the wild type protein suggesting that the folding stability of L44A is either enhanced or preserved.
The fourth chapter describes the synthesis and development of an adamantane-platinum conjugate (analog of carboplatin) hosted in beta-cyclodextrin. This inclusion complex formed by betaCD and the adamantane-platinum conjugate exhibited higher cytotoxicity towards neurobalstoma (SK-N-SH) cells than carboplatin. This approach provides versatility for enhancing drug properties via structural modification and targeted delivery using protein&ndashdrug conjugates
Prashar, Deepali, "Covalent Modification that Enhances Protein Properties and Functions and Folds Random Coil into Alpha-Helix" (2012). Chemistry - Dissertations. Paper 191.