Date of Award
5-2015
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Legacy Department
Bioengineering
Committee Chair/Advisor
Robert A Latour
Committee Member
Frank Alexis
Committee Member
Delphine Dean
Committee Member
Alexey Vertegel
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 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.
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