Computational simulation studies of substrate translocation pathways in bacterial membrane proteins

Abstract

Gram-negative bacterial membranes serve as a selective barrier to control the transport of nutrients and protect bacterial cells from harmful agents such as antibiotics and detergents. In order to mediate the regulation of cellular functions, bacterial membranes contain membrane proteins that are involved in the transport of ions, small molecules and macromolecules across the membrane layers. Many of these membrane proteins are known to be targets of antibiotics or to show specificities to certain substrates, and thus their structure, function, and dynamic behaviour must be thoroughly understood to be able to design new antibiotics. This thesis presents the use of computational simulations and high-resolution structures of specific membrane proteins provided from experimental collaborators to elucidate their substrate transport mechanism on a molecular level. Molecular Dynamics (MD) simulations, ranging from coarse-grained (CG) to all-atom (AA) models, were performed on selected membrane proteins: Either as an individual protein embedded in a lipid bilayer mimicking the inner or outer membrane of Gram-negative bacteria, or as a more complex system spanning across both membranes. My thesis developed from simple to more complicated models and techniques. My first study involved CG and AA models of the Mla protein embedded in a bacterial inner membrane (IM). Through collaboration with the Bergeron group at King's College London, I was able to provide the first molecular dynamics simulations of the MlaBDEF complex in an IM environment. In my next study, I investigated the transport mechanism of a hydrophobic transporter, TodX, in collaboration with the group of Bert van den Berg (Newcastle, UK). I performed free energy calculations using the umbrella sampling technique to uncover the energy barriers along our proposed transport pathway. Our simulations data presented indicate that TodX mediates substrate transport through a lateral opening in the protein. In my third study, I constructed an all-atom model of the Heme Acquisition System (HAS) complex embedded in two membranes and performed MD simulations to gain insight into the dynamic interplay between a TonB-dependent transporters (TBDT) and a TonB paralog. This project was a collaborative effort with the Izadi structural biology group in France. Finally, I built CG models of outer membrane vesicles (OMVs) and performed MD simulations to observe how the unsaturated lipid composition of the OMVs affects their shape preservation.

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ORCID for Jeremy Frey: orcid.org/0000-0003-0842-4302

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