Graduation Year
2023
Document Type
Thesis
Degree
M.S.
Degree Name
Master of Science (M.S.)
Degree Granting Department
Chemistry
Major Professor
Ioannis Gelis, Ph.D.
Committee Member
Jianfeng Cai, Ph.D.
Committee Member
David Merkler, Ph.D.
Keywords
protein folding, refolding/unfolding, guanidinium chloride denaturation, proline isomerization
Abstract
The conformational changes that occur during the folding of a protein is an intensely researched area because of the impact that it has on human health and cellular functions. Protein stability is crucial in the context of protein misfolding and aggregation due to its implications on misfolding diseases such as amyloid fibril degenerative diseases (Alzheimer’s, Parkinson’s). By examining the kinetics of protein folding, we can gain valuable information about the folding mechanism and help us identify potential targets for many of the protein misfolding diseases.
In this study, we present the folding and unfolding kinetics of TEM-1 β-lactamase from Escherichia coli over a range of denaturant concentrations, implementing fluorescence spectroscopy as the tool for characterizing the changes in folding state, and then fitting the time-dependent measurements with equations that describe the folding and unfolding phases. The kinetics is reported as a chevron plot and we discuss further the different studies that were done to understand the folding mechanism of TEM-1 β-lactamase.
The chevron plot indicates a midpoint denaturant concentration of 1.0 M. The refolding kinetics are biphasic at lower denaturant concentrations and are attributed to the final two slow phases of folding of TEM-1.
These findings and methods can be used as a benchmark protocol to determine the folding kinetics of related proteins. Additionally, we imply the utilization of rapid mixing methods to determine the characteristics of the intermediate formed at the early stages of folding. This can provide insight for understanding protein structure-function relationships and the importance of kinetics in terms of stability and structure.
Scholar Commons Citation
Dhananjayan, Dhanya, "Protein Folding Kinetics Analysis Using Fluorescence Spectroscopy" (2023). USF Tampa Graduate Theses and Dissertations.
https://digitalcommons.usf.edu/etd/9964
