Date of Award
Doctor of Philosophy (PhD)
Lesley H. Greene
Proteins are the fundamental building blocks of all living organisms. They are critical in the proper function of virtually all cellular processes and without them biological life would be impossible. Since their discovery, understanding the transition of DNA to functional protein has been separated into two distinct areas of research; 1) how are they synthesized from genetic material and 2) once formed how do they fold into their native tertiary and quaternary structures. Understanding how genetic material encodes the primary structure of amino acids was the basis of the first-half of the genetic code. This in part sparked a technological race to solve the sequence of whole genomes, leading to the human genome project and its completion in 2006. The second-half of the genetic code is known as the 'protein folding problem,' which is the primary focus of this work. This problem is focused on understanding the fundamental features that dictate how the primary structure of amino acids transition along a thermodynamic and kinetic pathway into a functionally folded native-state.
The protein folding problem is a multifaceted and interdisciplinary area of research. Two key avenues of investigation include the folding of a protein into its native structure and the second is misfolding into an amyloid fibril structure. In the first-half of this work, we investigated the folding of the BI domain of the Streptococcal immunoglobulin-binding domain of protein G (GB1) as our model system. Using bioinformatics approaches we investigated the origin and evolution of GB1. We also elucidated a group of conserved residues and a network of long-range interactions which we propose are key determinants in dictating the stability, folding and native structure of the protein. GB1 was initially characterized using a combination of biophysical and high-resolution techniques such as stopped-flow and equilibrium fluorescence, circular dichroism and nuclear magnetic resonance spectroscopy. A microbial system was developed to facilitate testing the role of conserved long-range interactions through site-specific 13C-labeling of tryptophan and phenylalanine. In the second-half, we investigated the transition of the Fas-associated death domain, an all α-helical Greek-key protein, into a misfolded amyloid-like state using additional techniques such as transmission electron and atomic force microscopy. From this work we were able to determine the extreme non-physiological conditions that would be required to allow for this transition. This result supports the 'generic amyloid hypothesis' that proposes that all proteins have the ability to form this alternative structure. In summary, this body of research has contributed to further advancing our understanding of the protein folding problem and laid the foundation for future atomic-level resolution studies.
Collins, Jason C..
"Investigation Into Protein Folding and Misfolding"
(2015). Doctor of Philosophy (PhD), dissertation, , Old Dominion University, DOI: 10.25777/gzq8-8k18