Doctor of Philosophy
Proteins perform various biological functions, e.g., as enzymes or transporters. In addition to naturally occurring proteins, the use of protein therapeutic drugs for treating cancer and other diseases is a rapidly growing area. A thorough biophysical characterization of proteins and protein therapeutics opens the door to a more comprehensive understanding of their role in health and disease. This dissertation aims to expand the capabilities of an existing technique (Hydrogen Deuterium Exchange Mass Spectrometry, HDX-MS), which is widely used for probing protein structure and dynamics. Conventionally, HDX-MS experiments are performed as a function of labelling time. Here we aim to establish temperature as a complementary variable. Our goal was to unravel the interplay between thermally induced protein dynamic motions, unfolding, and aggregation.
Chapter 2 examined the effects of protein heating, using myoglobin (Mb) as model system. MS was used to track deuterium uptake in response to increasing temperature at various labelling time points. The resulting data were captured using a comprehensive temperature- and time-dependent HDX data analysis framework. The HDX trends were dissected into contributions from “chemical” labelling, as well as local and global protein dynamics. Experimental profiles started with shallow slopes and showed a sharp increase close to the melting temperature. Our analysis revealed that local dynamics dominate at low temperatures, while global events become prevalent closer to the melting point.
Chapter 3 studied the mechanism of thermally induced Mb aggregation. Upon heating, Mb produced amorphous aggregates. The extent of aggregation was measured by centrifugation and UV-Vis spectroscopy as a function of protein concentration, temperature, and time. From these data, we conclude that aggregation likely proceeds from globally unfolded proteins rather than from semi-unfolded species. The data obtained this way paved the way toward extensive molecular dynamics simulations of protein aggregation.
In Chapter 4, we tested the applicability of the thermodynamic framework developed in Chapter 2 to a monoclonal antibody (NISTmAb), representing a model system of a typical protein therapeutic. Differential scanning calorimetry revealed the presence of three successive melting points, reflecting the different stability of the CH2, CH3, and Fab regions. HDX-MS was performed to comprehensively characterize the conformational dynamics of NISTmAb as a function of time and temperature. Global analysis of the entire data set yielded insights into the enthalpic and entropic behavior of different segments. The unfolding of the Fab domain (which has the highest melting temperature) was found to be closely coupled to aggregation. In summary, we developed a method that provides in-depth information on the thermodynamic behavior of thermally stressed proteins based on HDX-MS experiments, and we demonstrated the applicability of this method to proteins of vastly different sizes and complexity.
Summary for Lay Audience
Proteins are essential biological molecules in every living organism. Proteins also play an important role as therapeutics. For example, insulin has been used for decades in diabetic patients, and antibody drug conjugates are increasingly being used for treating various types of cancer. Unfavorable solvent conditions (such as heat and extremes of pH) can cause protein unfolding and aggregation. Protein aggregation is also a common characteristic of many neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases. Pharmaceutical tests of new protein drugs routinely use thermal unfolding and aggregation assays to assess the stability and shelf life of therapeutic proteins. However, there remains an urgent need to develop new and improved methods for monitoring protein thermal stability. With this in mind, the aim of the current thesis was to develop a better understanding of thermally stressed proteins.
In Chapter 2, myoglobin, a small globular protein was used as model system. Myoglobin was incubated in heavy water, and mass spectrometry was used to track deuterium uptake in response to increasing temperature. We developed a novel model to interpret the temperature- and time-dependent deuterium uptake data. Our analysis revealed that local structural fluctuations dominate the protein behavior at low temperatures, while global unfolding/refolding became prevalent close to the melting point.
Chapter 3 studied the mechanism of thermally induced myoglobin aggregation. The extent of aggregation was measured by centrifugation and UV-Vis spectroscopy as a function of protein concentration, temperature, and time. From these data, we concluded aggregation proceeds from totally unfolded proteins rather than from semi-folded intermediates.
In Chapter 4, we tested the applicability of the thermodynamic framework developed in Chapter 2 to a monoclonal antibody, representing a model system of a therapeutic protein. Differential scanning calorimetry revealed that this multi-domain protein has three melting points. Incubation of the antibody in heavy water at different temperatures and time points, followed by mass spectrometry analysis, was conducted to comprehensively characterize the conformational dynamics of the protein. Our data yielded insights into the thermodynamic behavior of different protein segments. In summary, we developed a novel method that provides in-depth information on the thermodynamic behavior of thermally stressed proteins.
Nosrat Tajoddin, Nastaran, "Conformational Dynamics and Aggregation of Thermally Stressed Proteins Studied by Hydrogen/Deuterium Exchange Mass Spectrometry" (2022). Electronic Thesis and Dissertation Repository. 8949.