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Investigation Into the Effects of Pegylation on the Thermodynamic Stability of the WW Domain

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Investigation Into the Effects of Pegylation on the Thermodynamic Stability of the WW Domain Brigham Young University BYU ScholarsArchive Theses and Dissertations 2013-12-01 Investigation into the Effects of PEGylation on the Thermodynamic Stability of the WW Domain Sam S. Matthews Brigham Young University - Provo Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Biochemistry Commons, and the Chemistry Commons BYU ScholarsArchive Citation Matthews, Sam S., "Investigation into the Effects of PEGylation on the Thermodynamic Stability of the WW Domain" (2013). Theses and Dissertations. 4280. https://scholarsarchive.byu.edu/etd/4280 This Thesis is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Investigation into the Effects of PEGylation on the Thermodynamic Stability of the WW Domain Sam Scowcroft Matthews A thesis submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Master of Science Joshua L. Price, Chair Paul B. Savage Steven W. Graves Department of Chemistry and Biochemistry Brigham Young University December 2013 Copyright © 2013 Sam Scowcroft Matthews All Rights Reserved ABSTRACT Investigation into the Effects of PEGylation on the Thermodynamic Stability of the WW Domain Sam Scowcroft Matthews Department of Chemistry and Biochemistry BYU Master of Science The covalent attachment of poly(ethylene glycol) (PEG) to a protein surface (known as PEGylation), has been demonstrated to increase the serum half-life of therapeutic proteins by reducing kidney clearance and immunogenicity and by protecting against proteolysis. Theses beneficial effects could be further enhanced if PEGylation consistently increased protein conformational stability (i.e. the difference in free energy between the folded and unfolded states). However, the effects of PEGylation on protein conformational stability are unpredictable; PEGylation has been reported to increase, decrease, or have no effect on the conformational stability of medicinal proteins. This thesis details the results of two studies aimed at discovering the structural determinants which influence the thermodynamic impact of PEGylation on the WW domain, a small model protein. Chapter 1 is a brief introduction to protein therapeutics and protein PEGylation. Chapter 2 describes a study which demonstrates that the thermodynamic impact of PEGylation is strongly dependent on the site to which PEG is conjugated. The studies described in Chapter 3 elaborate on this site dependence, and demonstrate that PEG stabilizes the WW domain through interactions with the surface of the folded peptide, and that two factors – the orientation of the PEG chain (relative to the protein surface) and the identity of nearby side chains – play a critical role in determining the thermodynamic impact of PEGylation. Keywords: PEGylation, Therapeutic Proteins, Thermodynamic Stability, Circular Dichroism, β-sheet, D-Amino Acids ACKNOWLEDGEMENTS Thanks to my advisor, Josh Price. From the beginning of my graduate program, he encouraged me to “own” my projects, and has allowed me to play an active role in the design and implementation of every project I was involved in during these past 2 years. Thanks also to my lab-mates, with whom I collaborated on many of the experiments presented in this thesis. Thanks specifically to Paul Lawrence, with whom I shared the experience of being Dr. Price’s first graduate student. Whenever something went wrong, he was the first person I turned to. Many of the results in Chapter 1 were obtained with his help. Thanks also to Brijesh Pandey, our post-doc, who, among other things, agreed to watch over my shoulder and tell me what I was doing wrong when none of my peptide syntheses worked properly. Finally, thanks to all the undergraduate students who helped in the synthesis, purification, and characterization of many of the peptides used in my research. To Mindy, Chad, Mason, Ryan, Minnie, and Cameron, thank you. Table of Contents Chapter 1: Introduction to Protein Therapeutics and PEGylation ·························································· 1 1.1 Protein Therapeutics ······················································································································· 2 1.1.1 Insulin ········································································································································· 2 1.1.2 Advantages of Protein Therapeutics ·························································································· 2 1.1.3 Challenges for Protein Therapeutics ·························································································· 3 1.2 Protein Folding ································································································································ 3 1.3 Strategies to Improve Protein Pharmacokinetics ············································································ 4 1.3.1 Sequence Modification ·············································································································· 4 1.3.2 Glycosylation ······························································································································ 5 1.3.3 Non-Natural Modifications to Protein Surfaces ········································································· 6 1.4 PEGylation ······································································································································· 6 1.5 Effects of PEGylation on Serum Half-Life ························································································ 7 1.5.1 PEGylation Reduces Immunogenic Response ············································································ 7 1.5.2 PEGylation Decreases Kidney Clearance ···················································································· 8 1.5.3 PEGylation Protects Against Proteases ···················································································· 10 1.6 Methods of PEGylation·················································································································· 11 1.7 Drawbacks to PEGylation ·············································································································· 14 1.7.1 The Effect of PEGylation on Protein Thermodynamic Stability is Difficult to Predict. ············· 16 1.8 Current Research into the Thermodynamic Consequences of PEGylation ···································· 17 1.8.1 PEG May Stabilize Proteins by Decreasing the Solvent Accessible Surface Area of the Folded State ········································································································································· 17 1.8.2 PEG May Act to Decrease the Conformational Dynamics of the Protein ································· 18 1.8.3 Previous Work in the Price Lab ································································································ 19 1.9 References ····································································································································· 21 iv Chapter 2: The Thermodynamic Consequences of PEGylation Depend on the Site of Poly(Ethylene Glycol) Attachment ············································································································ 28 2.1 Introduction ··································································································································· 29 2.2 Results and Discussion ·················································································································· 31 2.2.1 Initial PEG Scan ························································································································· 31 2.2.2 PEGylation of Reverse Turns ···································································································· 36 2.2.3 PEGylation of a β-Strand ·········································································································· 37 2.3 Conclusions ···································································································································· 37 2.4 Supporting Information ················································································································· 40 2.4.1 Protein Synthesis ······················································································································ 40 2.4.2 Purification and Characterization ····························································································· 41 2.4.3 ESI-TOF ····································································································································· 42 2.4.4 HPLC ········································································································································· 52 2.4.5 Analysis of Thermal and Kinetic Parameters ············································································ 61 2.4.6 Circular Dichroism Spectropolarimetry ···················································································· 61 2.4.7 Laser Temperature Jump Experiments ···················································································· 61 2.4.8
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