Date of Graduation

12-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Biological Sciences

Advisor/Mentor

Mahmoud Moradi

Committee Member

Colin Heyes

Second Committee Member

Suresh Thallapuranam

Third Committee Member

Yong Wang

Keywords

Molecular dynamics, Transition pathways, Superfamily Transporters

Abstract

Molecular dynamics (MD) simulations are routinely used to study the dynamics of proteins. However, conventional MD limited to the sampling of local conformational changes as the functionally important conformational transitions of proteins often extend beyond the timescales of the simulations employed, for example, membrane transport proteins. We have determined the combination of multiple MD based techniques that allows for a rigorous characterization of energetics and kinetics of large-scale conformational changes in membrane proteins. The methodology is based on biased, nonequilibrium collective variable based simulations including nonequilibrium pulling, string method with swarms of trajectories, bias-exchange umbrella sampling, and rate estimation techniques. Severe acute respiratory syndrome (SARS) coronaviruses 1 and 2 (SARS-CoV-1 and SARS-CoV-2) spike proteins undergo conformational changes in the receptor binding domain (RBD) in order to bind to the host cell angiotensin converting enzyme 2 (ACE2). However, the mechanistic details that describe the large-scale conformational changes associated with spike protein activation or deactivation are still somewhat unknown. We have employed a simulation strategy thatwe have shown to be successful in characterizing the confromational changes in membrane proteins and have elucidated the transition pathways of protein deactivation for the SARS-CoV-1 (CoV-1) and SARS-CoV-2 (CoV-2) prefusion spike proteins. The resulting transition pathways were shown to be unique to both CoV-1 and CoV-2 . A number of key residues that form various interdomain salt-bridges have been identified. We have also constructed the free energy profiles along the transition pathways for both CoV-1 and CoV-2. The CoV-2 spike protein must overcome larger free energy barriers to undergo conformational changes towards protein activation or deactivation, when compared to CoV-1. We have performed a variety of biased and unbiased MD simulations on the bacterial proton-coupled oligopeptide transporter GkPOT, glucose transporter 1 (GluT1), and glycerol-3-phosphate transporter (GlpT) to compare the similarities and differences of the conformational dynamics of three different classes of MFS transporters. Here we have simulated the apo protein in an explicit membrane environment. Our results suggest a very similar conformational transition involving interbundle salt-bridge formation/disruption coupled with the orientation changes of transmembrane (TM) helices, specifically H1/H7 and H5/H11, resulting in an alternation in the accessibility of water at the cyto- and periplasmic gates.

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