Date of Award

Winter 2023

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

First Advisor

Krisztina Varga

Second Advisor

Richard H Cote

Third Advisor

Feixia Chu

Abstract

In this work, I have explored basic questions related to protein structure, function, and stability, which are critical for understanding the fundamental mechanisms of biology. The dissertation is divided into four chapters, which are different topics I have worked on. In Chapters 1 and 2, I will discuss my work on two different antifreeze proteins, in Chapter 3 my work on the main protease from SARS-CoV-2 is presented, while Chapter 4 details the characterization of protein stability of four B. subtilis proteins. While the projects are distinct from one another, they all contribute to deciphering the molecular basis of life processes and advancing our understanding of protein biochemistry. The topics are summarized here:

Chapter 1 presents the findings of the study investigating the correlation between structural stability and activity of the antifreeze protein from a desert beetle (Anatolica polita), Trx-ApAFP752. The Varga group has previously structurally characterized ApAFP752 using nuclear magnetic resonance (NMR) spectroscopy, that revealed its unique fold (i.e., b-helix), commonly found among insect AFPs. Here we demonstrated that Trx-ApAFP752 not only has high thermal stability but can provide cryoprotection during freeze/thaw for a freeze sensitive enzyme, lactose dehydrogenase (LDH).

Chapter 2 presents the findings of the study investigating secondary structure and cryoprotection of a truncated antifreeze protein from a broadleaf shrub, Ammopiptanthus nanus, AnAFP-AKseg. Based on the sequence homology, this protein can be classified as a dehydrin-like antifreeze protein. We have shown that AnAFP-AKseg can preserve LDH activity after freeze/thaw.

Chapter 3 presents the experimental findings of the study conducted in collaboration with the Vashisth group (University of New Hampshire). The study investigated the inhibition of the main protease from SARS-CoV-2, which is an important drug target for the treatment of COVID-19. In our study, we selected two thiadiazolidinone compounds (i.e., CCG-50014 and CCG-203769) that share structural similarities with a known main protease inhibitor, tideglusib. Our experimental inhibition assays and the computational studies demonstrated that CCG-50014 is a potent covalent inhibitor for SARS-CoV-2 MPro. Our experimental results indicated that CCG-203769 inhibits MPro as well but with lower potency.

Chapter 4 discusses results of a thermal stability study of four selected proteins form a model organism, B. subtilis. We assessed their thermal stability using circular dichroism spectroscopy that allowed us to calculate the thermodynamic parameters describing unfolding of these proteins. The Varga group is currently performing additional analyses of other B. subtilis to investigate this correlation.

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