How Does Varying Sizes and Concentrations of Poly(Ethylene Glycol) Affect the Conformation and Function of the Enzyme Escherichia Coli Prolyl-Trna Synthetase?

Color poster with text, charts, images, and graphs.Poly(ethylene glycol) (PEG) is a biocompatible, hydrophilic, flexible, non-toxic polyether commonly used in proteomics because it is widely regarded to be a biologically inert molecule. This includes PEG2000 (molecular weight of 2000g/mol), which is a component in the Pfizer-BioNTech and Moderna COVID-19 vaccines. However, recent studies suggest that PEG may impact protein function, depending largely on the molecular weight of the PEG. In this study, we observe the effects of PEG on the function and conformation of enzyme prolyl-tRNA synthetase of Escherichia coli (Ec ProRS). ProRS catalyzes the covalent attachment of proline to tRNAPro and thus is essential for protein biosynthesis. The molecular mechanism of PEG-protein interactions has been probed using enzyme kinetics, fluorescence spectroscopy, and molecular modeling. Herein, we present the results of our study.National Institute of Health (Grant#:1R15GM117510); University of Wisconsin--Eau Claire Office of Research and Sponsored Program

Molecular Dynamics Simulation Studies to Probe the Impact of Oxidative Stress on the Binding of SARS-CoV-2 Spike Protein to Angiotensin-Converting Enzyme 2

Color poster with text, images, charts, and graphs.The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein as well as the human cell surface receptor angiotensin-converting enzyme II (ACE2) contain several cysteine residues. These cysteine residues exist either in the form of disulfide bridges (oxidized) or as thiols (reduced). The thiol-to-disulfide equilibrium is shifted to the right when there are excess reactive oxygen species (ROS) in the body, which is referred to as oxidative stress. It has been shown that certain preexisting conditions, associated with oxidative stress, such as diabetes, obesity, heart conditions, and age can put individuals at a higher risk of contracting COVID-19.  Therefore, we hypothesized that the oxidized state of these proteins may have an impact on the binding of the virus protein to the receptor. To test our hypothesis, we used molecular dynamics simulations to study the interacting residues at the binding interface of the complex formed by the receptor-binding domain of SARS-CoV-2 and the peptidase domain of ACE2. Four complexes of ACE2 and SARS-CoV-2 in different redox states were generated by either preserving the disulfides or reducing them to thiols. The preliminary data and findings of this study will be presented.National Conference on Undergraduate Research (NCUR); National Institute of Health; University of Wisconsin--Eau Claire Office of Research and Sponsored Program

Role of a Conserved Disulfide on the Interactions Between Severe Acute Respiratory Syndrome Coronavirus 2 and Angiotensin-Converting Enzyme 2

Color poster with text, charts, and images.Coronaviruses are large, enveloped, positive strand RNA viruses capable of infecting a large array of mammalian and avian species that possess densely glycosylated spike-shaped proteins on their surfaces giving them the appearance of crowns under electron microscope, hence their name. The receptor binding domain (RBD) of the spike protein specifically recognizes and binds to the extracellular peptidase domain of the human angiotensin-converting enzyme 2 (ACE2) with high affinity. There is some evidence to suggest that the entry of viral glycoprotein is affected by the thiol-disulfide balance on the cell surface and disrupting this balance can prevent the virus from being able to infect the host cell. Both the RBD of the spike protein and ACE2 contain several cystine residues, and the existence of several disulfide bridges within them has been established when the species are under oxidative stress. It has also been established that the complete reduction of these disulfide bridges to sulfhydryl groups completely impairs the ability of the RBD to bind to ACE2. However, it is still unknown how each individual disulfide bridge in these proteins impacts the binding. In this study, in a hope to gain an insight into a possible mechanism of disrupting the virus’s life cycle, the disulfide bridge between residues C344 and C361 in ACE2 were probed using molecular dynamics simulations. Results indicated that the removal of the investigated disulfide bridge is insufficient to disable binding between the proteins.University of Wisconsin--Eau Claire Office of Research and Sponsored Program