Structure-Function-Dynamics Relationships of Protein-Polymer Conjugates: Improving Activity and Stability in Non-Native Environments
Creators: name: Baker, Stefanie
Publisher: name: Carnegie Mellon University
Year: 2019
Subjects: subject: 90399 Biomedical Engineering not elsewhere classified
subject: FOS: Medical engineering
subject: FOS: Medical engineering

description: Evolution has optimized the activities and selectivities of proteins at the cost of stability. Certain organisms, known as extremophiles, contain specially adapted proteins that thrive in extreme conditions such as high temperature or high salt; however, the majority of proteins are only able to survive in moderate environments. Thus, protein engineers have long been interested in modifying proteins to increase their stabilities in non-native environments. One method of protein modification is attaching synthetic polymers to the protein surface. There are two main approaches to create these protein-polymer conjugates. Pre-synthesized polymers can be grafted to the protein surface or polymers can be grown from a protein macro-initiator monomer by monomer to a desired length using a controlled radical polymerization technique. Covalently attaching polymers to a protein alters the protein’s physicochemical properties (size, bioactivity, stability, solubility), but not always in a predictable way. The key to rationally designing protein-polymer conjugates that display a desired property is to understand the underlying protein-polymer interfacial interactions that drive changes in protein function. In this work, the structure-function-dynamics relationships of grafted from protein-polymer conjugates were investigated using experimental and computational methods in order to improve their activities, stabilities, and solubilities in non-native environments. In Chapter 2, various charged polymers of varying lengths were grown from α-chymotrypsin. Different polymer types altered enzyme bioactivity by changing substrate affinity. Additionally, a mechanism was developed to explain how polymers stabilized proteins at low pH. Zwitterionic and positively charged polymers prevented protein unfolding and assisted in protein refolding to increase stability at pH 1. In Chapter 3, the effect of atom-transfer radical polymerization (ATRP) initiator structure on protein and protein-polymer conjugates was determined. Positively charged ATRP initiators restored the native surface charge of proteins, when targeting surface accessible amino groups, which increased the protein-initiator and subsequent protein-polymer conjugate activities and stabilities at low pH and high temperature. In Chapter 4, the solubility of lysozyme was predictably tuned by polymer conjugation. Zwitterionic polymers increased lysozyme solubility and prevented precipitation in fully saturated ammonium sulfate salt with maintained bioactivity while amphiphilic polymers decreased solubility. Zwitterionic polymers displayed an anti-polyelectrolyte effect in increasing salt which increased the number of hydration layers around the conjugate to increase solubility. Amphiphilic polymers collapsed around the protein surface in increasing salt concentrations and decreased in hydration which promoted precipitation. The differences in solubilities were utilized to purify mixtures of native protein and protein-polymer conjugates into homogeneous components. In all of these Chapters, atomistic molecular dynamics simulations were employed to enhance the mechanistic understanding of protein-polymer conjugate structure-function-dynamics relationships. The knowledge gained through these combined studies helps to demystify how covalently attached polymers impact protein function which can lead to new applications of protein-polymer conjugates in therapeutic and biotechnological industries. <br>