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Title: Investigating protein properties via microfluidic techniques
Author: Zhang, Yuewen
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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Proteins are one of the most abundant biomacromolecules and play important roles in bioactivities within organisms, including key functions in DNA replication, immune system response, metabolic reactions and molecular transportation. Proteins fold into their unique compact three-dimensional structures to precisely perform their functions. Knowledge of proteins on their structures and interactions with other molecules or biomacromolecules is fundamental to understand the mechanism of many diseases. Thus, in this thesis, the focus is on studying the physical properties of proteins, and protein interactions using microfluidic techniques. Chapter 1 provides a brief introduction to protein stability and identification as well as their interactions. Then, conventional techniques for studying protein systems are reviewed. Moreover, the principles, designs and applications of microfluidic techniques are introduced. Chapter 2 demonstrates that a microfluidic diffusional sizing device can be employed to study protein interactions with small molecules by measuring the variation of hydrody- namic radius of bovine serum albumin (BSA) in aqueous solution as a function of pH. By simulating the behaviour of folded and unfolded BSA, the relative population of BSA in dif- ferent states can be calculated. In addition, the key residues that regulate the BSA unfolding process are predicted. Furthermore, I utilize a modified microfluidic diffusional sizing device to measure the hydrodynamic radius of Escherichia coli 70S ribosome and study its interactions with antibiotics, such as chloramphenicol. In Chapter 3, a microfluidic device for protein identification is designed to measure the physical parameters of proteins, including the fluorescence intensities of specific amino acids (tryptophan, tyrosine, lysine) and the hydrodynamic radius of proteins. Thus, proteins can be correctly identified in a single microfluidic device. The results have significant implications in development of effective and efficient techniques for precise protein identification in their native state. In Chapter 4, the interactions between a membrane protein (aquaporins) and a small regulator protein (calmodulin) are investigated by detecting the changes of their electrophoretic mobility and diffusivity in microfluidic devices. Then the effective charges of aquaporin0 and its complex with calmodulin are calculated. Finally, the selective binding between calmodulin and different types of aquaporins is investigated based on their relative binding affinities.
Supervisor: Knowles, Tuomas Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: microfluidics ; protein-protein interactions ; protein physical properties