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Development, Validation, and Application of Analytical Methods for Characterizing Adsorbed Protein Orientation, Conformation, An Clemson University TigerPrints All Dissertations Dissertations 5-2015 DEVELOPMENT, VALIDATION, AND APPLICATION OF ANALYTICAL METHODS FOR CHARACTERIZING ADSORBED PROTEIN ORIENTATION, CONFORMATION, AND BIOACTIVITY Aby Thyparambil Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_dissertations Part of the Biochemistry Commons, and the Biological and Chemical Physics Commons Recommended Citation Thyparambil, Aby, "DEVELOPMENT, VALIDATION, AND APPLICATION OF ANALYTICAL METHODS FOR CHARACTERIZING ADSORBED PROTEIN ORIENTATION, CONFORMATION, AND BIOACTIVITY" (2015). All Dissertations. 1512. https://tigerprints.clemson.edu/all_dissertations/1512 This Dissertation is brought to you for free and open access by the Dissertations at TigerPrints. It has been accepted for inclusion in All Dissertations by an authorized administrator of TigerPrints. For more information, please contact [email protected]. DEVELOPMENT, VALIDATION, AND APPLICATION OF ANALYTICAL METHODS FOR CHARACTERIZING ADSORBED PROTEIN ORIENTATION, CONFORMATION, AND BIOACTIVITY A Dissertation Presented to the Graduate School of Clemson University In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Bioengineering by Aby Abraham Thyparambil May 2015 Accepted by: Dr. Robert A. Latour, Committee Chair Dr. Alexey Vertegel Dr. Delphine Dean Dr. Frank Alexis ABSTRACT The structure and bioactivity of adsorbed proteins are tightly interrelated and play a key role in their interaction with the surrounding environment. These factors are of critical importance in many biotechnological applications. However, because the bioactive state of an adsorbed protein is a function of the orientation, conformation, and accessibility of its bioactive site(s), the isolated determination of just one or two of these factors will typically not be sufficient to understand the structure-function relationships of the adsorbed layer. Rather a combination of methods is needed to address each of these factors in a synergistic manner to provide a complementary dataset to characterize and understand the bioactive state of adsorbed protein. In this research, I describe and demonstrate the potential of a set of complementary methods: (a) circular dichroism spectropolarimetry to determine adsorption-induced changes in protein secondary structure, (b) amino-acid labeling/mass spectrometry to assess adsorbed protein orientation and tertiary structure by monitoring adsorption-induced changes in a residue’s solvent accessibility, and (c) bioactivity assays to assess adsorption-induced changes in a protein’s bioactivity. Subsequently, the developed techniques were applied to characterize: (a) the role of protein-protein interactions (PPI) in influencing the structure and activity of a protein during its layer formation, and (b) the influence of chemical excipients on the stability and potency of an adsorbed layer of protein. While the effect of PPI on the initial adsorbed configuration and bioactivity of a protein layer varied with the type of adsorbent surface and protein composition, the effects of chemical excipients on the stability and potency of an adsorbed protein layer primarily depended on its initial ii adsorbed configuration. From an evaluation of the structure-function relationship within these adsorbed layers, their bioactivity was found to reduce in direct proportion to the disruption in protein structure in majority of the systems studied. Although, the presented techniques do have the limitation of being low in resolution, the techniques developed in this study do provide insights into the molecular processes influencing the structure- function relationships of adsorbed protein that were previously unknown. iii DEDICATION To my ‘Rô’eh’, and one of the finest teacher I know. iv ACKNOWLEDGMENTS First and foremost I offer my sincerest gratitude to my advisor, Dr. Robert Latour, who has supported me throughout my PhD studies with his patience and knowledge whilst allowing me the room to work in most of my own way. In this equal note, I would also like to express my sincere gratitude to Dr. Yang Wei as well, for his mentoring throughout this doctoral work. I do owe a lot of my academic accomplishments to the sincere efforts from both of them. Sincere thanks are due to my committee members, Dr. Alexey Vertegel, Dr. Delphine Dean, and Dr. Frank Alexis for their accommodative nature. I would also like to thank the past and present members of Biomolecular Interactions and Biomolecular Modeling laboratories, Dr. Yonnie Wu, (Auburn University) and Dr. Guzeliya Korneva, for their support. Likewise, I sincerely thank other faculty and support staff at the Department of Bioengineering, for all the assistances provided throughout my time at Clemson. Moreover, I sincerely thank and acknowledge the facility and funding supports from DTRA (Grant no. HDTRA1-10-1-0028), NIH NIBIB (grant # EB002027) and NIH Grants (5P20RR021949-04 and 8P20GM103444-04). On a closing note, I additionally acknowledge the immense scientific help provided by the blogs of Dr. Martin Chaplin (London South Bank University) and Philip Ball. Special thanks to my friends and their families, for making the time in Clemson so memorable and eventful. Lastly, I am sincerely and most grateful to my parents, brothers, and my dear wife for their patience, prayer, support, encouragement and inspiration provided throughout my doctoral years. v TABLE OF CONTENTS Page TITLE PAGE .................................................................................................................... i ABSTRACT ..................................................................................................................... ii DEDICATION ................................................................................................................ iv ACKNOWLEDGMENTS ............................................................................................... v LIST OF TABLES ................................................................................................... ix- xii LIST OF FIGURES .............................................................................................. xiii- xvi CHAPTER I. INTRODUCTION .......................................................................................... 1– 4 II. BACKGROUND ......................................................................................... 5 – 78 Introduction .............................................................................................. 5 Methods to characterize the molecular processes influencing the bioactivity of adsorbed/immobilized protein layer .................................. 9 Factors influencing the bioactive state of adsorbed/immobilized protein layer ........................................................................................... 26 Molecular mechanisms involved in the interaction of an aqueous solution of excipient with an adsorbed/immobilized protein layer ........ 46 Chapter summary and Conclusion ......................................................... 55 References .............................................................................................. 58 III. SPECIFIC AIMS ...................................................................................... 79 – 81 Aim 1 ..................................................................................................... 79 Aim 2 ..................................................................................................... 80 Aim 3 ..................................................................................................... 81 IV. EXPERIMENTAL CHARACTERIZATION OF ADSORBED PROTEIN ORIENTATION, CONFORMATION, AND BIOACTIVITY ........................... 82 – 112 Introduction ............................................................................................ 82 Experimental set-up and Methodology .................................................. 87 vi Table of Contents (Continued) Page Chapter summary and Conclusion ....................................................... 105 References ............................................................................................ 107 V. QUANTIFICATION OF THE INFLUENCE OF PROTEIN-PROTEIN INTERACTIONS ON ADSORBED PROTEIN STRUCTURE AND BIOACTIVITY .................................................................................... 113 – 158 Introduction .......................................................................................... 113 Experimental set-up and Methodology ................................................ 116 Results and Discussion ........................................................................ 128 Chapter summary and Conclusion ....................................................... 153 References ............................................................................................ 155 VI. DETERMINATION OF ORIENTATION AND ADSORPTION-INDUCED CHANGES IN THE TERTIARY STRUCTURE OF PROTEINS ON MATERIAL SURFACES BY CHEMICAL MODIFICATION AND PEPTIDE MAPPING ............................................................................. 159 – 193 Introduction .......................................................................................... 159 Experimental set-up and Methodology ................................................ 162 Results and Discussion ........................................................................ 171 Chapter summary and Conclusion ......................................................
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