Date of Graduation

8-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry (PhD)

Degree Level

Graduate

Department

Chemistry & Biochemistry

Advisor/Mentor

Lay, Jackson O. Jr.

Committee Member

Moradi, Mahmoud

Second Committee Member

Adams, Paul

Third Committee Member

Thallapuranam, Suresh

Keywords

HDX-ESI-MS; Human acidic Fibroblast Growth Factor; Peptic peptide stability; Protein Equilibrium Population Snapshot; Protein stability; Staphylococcal nuclease

Abstract

This dissertation presents a detailed investigation into the applications of Protein Equilibrium Population Snapshot H/D Exchange Electrospray Ionization Mass Spectrometry (PEPS-H/D exchange or PEPS) method. This method is based on protein equilibrium unfolding, and its fundamental capability is to capture a "snapshot" of the protein's state, allowing us to monitor the ratio of the unfolded to folded populations at different chemical denaturant concentrations. First, to increase the accuracy of PEPS data analysis for assessing protein stability, the traditional linear extrapolation method (LEM) was replaced. Instead of using LEM, which calculates free energy change based on the peak ratio of unfolded and folded population mass-to-charge ratios (m/z), the use of a weighted average of both m/z values was adopted to determine the molecular weight. The molecular weights obtained from all the denaturant concentrations are then fitted to a theoretical (calculated) molecular weight using a non-linear regression tool. This optimized PEPS was then utilized to measure the stability of staphylococcal nuclease (STW) and human acidic fibroblast growth factor (hFGF1) under chemical denaturing conditions. For STW, PEPS experiments yielded reproducible values for free energy change (ΔG), linear denaturation constant (m-value), and concentration at which half of the protein is denatured (Cm), consistent with literature values from other techniques. Regional unfolding dynamics were mapped by peptide-level H/D exchange after pepsin digestion of the intact protein, revealing a correlation between secondary structure content and susceptibility to chemical denaturation. The study extended the promising results from STW analysis to investigate the stability of wild-type hFGF1 and its R136D variant. The R136D mutation enhanced protein stability, indicated by an increased Cm. PEPS data for hFGF1 agreed with theoretical solvent accessibility after correcting for back-exchange. Furthermore, the influence of pH and salt concentration on hFGF1 stability was systematically evaluated using intrinsic fluorescence spectroscopy. Increased pH and ammonium sulfate concentration increased Cm values, suggesting improved stability. However, the ion pairing interactions between guanidinium and sulfate ions, known to stabilize proteins, may have contributed to increased Cm values. Also, the distinct patterns in hFGF1 unfolding in response to extreme pH and the addition of various concentrations of ammonium sulfate provided insights into possible stable intermediates in the protein. This work demonstrates the utility of PEPS techniques, particularly for determining protein stability parameters and regional unfolding dynamics. It also provided insight into the effect of mutation and environmental conditions on the electrostatic interactions within hFGF1. This dissertation demonstrates that PEPS is consistent with other methods, underscoring its potential for wider proteomics, structural biology, and biomarker discovery applications. This work contributes to understanding the relationships between protein structure and function and the environmental factors that influence protein stability.

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