Structural and functional studies on Gram-negative bacteria lipid transport and modification proteins

Zhang, Zhengyu (2017) Structural and functional studies on Gram-negative bacteria lipid transport and modification proteins. Doctoral thesis, University of East Anglia.

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Gram-negative bacteria contain a double membrane system which is made up of the inner membrane (IM) and the outer membrane (OM). The OM, the asymmetric membrane, is an important contributor towards resistance against various toxic molecules which provides extensive protection of cell viability. The OM consists of lipopolysaccharides (LPS) at the outer leaflet and glycerophospholipid (GPL) at the inner leaflet. LPSisa large amphipathic moleculethatis essential for most Gram-negative bacteria and is composed of three moieties: lipid A, core oligosaccharide and O-antigen. Precursors of LPS, the rough LPS and O-antigen units, are synthesized at the inner leaflet of the IM and are assembled into mature LPS at the outer leaflet of the IM. Seven proteins, lipopolysaccharide transporters (LptA-G), form a periplasmic bridge and transport mature LPS from the IM tothe outer leaflet of the OM, where LPS functions as an impermeable barrier. The inner leaflet of the OM is also an obligate componentfor the stability and functionality of OM. GPL molecules consist of phosphatidylethanolamine (PE), phosphatidylglycerols (PG) and cardiolipin (CL) in an approximately 75: 20: 5 ratio in most bacterial membranes. The biogenesis and transport of LPS and GPL are often the highlights of novel drug development; however little is known about the mechanisms of the actionsof the proteins involved in these processes.

Protein X-ray crystallography is a technique that determinesthe molecular structure of a protein or a protein complex from the diffraction data of a protein crystal generated underX-ray beam. Knowing a protein’s molecular structure provides us with the informationwhich may include possible molecular motion and conformational changes or interactions between residues, substrates and domains. These important details may provide hints into how to perform further studies on broad-ranged specific targets.

This thesis includes four Chapters related to the X-ray crystal determination of proteins involved in biosynthesis, modificationand transport of LPS and the transport of CL. In Chapter 1 I have included the successful cloning, expression and structure determination of the ABC transporter LptB2FG, which is responsible for LPS extraction from the inner membrane. Also, the corresponding in vivoand in vitroassays are reported to support the LPS extraction and transportation functionality of LptB2FG. Chapter 2 contains the successful expression of glycosyl transferase WaaB and the determination of its structure. Mutagenesis and enzyme activity assays have also been performed to determine the galactosyl transferase activity site of WaaB(work by Gareth James Asheworth). Chapter 3 describesthe successful cloning and structural determination of LapB, which may controlan intermediated step of lipid A biosynthesis via balancing the level of LpxC & FtsH, Finally, Chapter 4 reveals the protein expression and structural determination of the globular domain of PbgA. PbgA is regarded as the cardiolipin transportation protein and we have also performed in vivo mutagenesis functional assays to identify the important residues for its functionality.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User: Users 9280 not found.
Date Deposited: 22 Mar 2018 12:51
Last Modified: 31 Mar 2019 01:00


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