Structural and functional characterisation of novel bacterial Toll/Interleukin-1 receptor (TIR)-like proteins.

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Title: Structural and functional characterisation of novel bacterial Toll/Interleukin-1 receptor (TIR)-like proteins.
Author(s): Rana, Rohini R
Item Type: Thesis or dissertation
Abstract: The extracellular domain of Toll-like receptors (TLRs) recognises highly specific pathogen-associated molecular patterns in innate immunity. This causes molecular rearrangement of the intracellular Toll/Interleukin-1 (TIR) domains of the TLRs allowing recruitment of downstream adaptor proteins via heterotypic TIR-TIR protein interactions. This in turn initiates a signalling cascade leading to proinflammatory immune responses. Recent work has indicated that TIR-like proteins (TLPs)from pathogenic bacteria contain TIR domains and interfere with host TLR signalling. Bacterial TLPs are suggested to bind to the TIR domains of host TLRs and/or adaptor proteins, thereby inhibiting intracellular signalling. This project focuses on characterisation of the TLPs from the highly pathogenic bacteria Yersinia pestis (YpTLP) and Burkholderia pseudomallei (BpTLP). The aim of the project was to produce soluble, pure and stable TLPs of yields suitable for functional and structural studies. Bacterial TLPs were expressed in E. coli. Expression of the full-length YpTLP and BpTLP using pWaldo-GFPe yielded 2.78 mg/l and 2.52 mg/l respectively. Protein purification steps were complicated by protein precipitation, possible degradation and misfolding. Subsequent efforts focused on the expression of the TIR only domain/homologue regions: YpTIR and BpTIR. Soluble YpTIR and BpTIR were expressed using pET26b. GST pull down assays indicated positive interactions between His-tagged YpTIR/BpTIR and GST-tagged human MyD88-TIR, a major adaptor protein, revealing MyD88 as a potential target of the bacterial TLPs. However expression yields of pure protein were too low to allow further studies. YpTIR and BpTIR were then expressed as fusions with an N-terminal GB1 tag using GEV2. Subsequent purification produced highly pure GB1-YpTIR (5 mg/l). NMR analysis indicated that the protein was folded and likely to be in a dimeric form, a finding confirmed by gel filtration. Native and derivative crystals of YpTIR were obtained, and native diffraction datasets collected to 2.95 A. Further work includes obtaining the phase information. It is anticipated that the crystal structure of YpTIR will provide insight into the molecular basis of TIR signalling and evidence of evolutionary conservation among TIR domains.
Publication Date: Jun-2010
Date Awarded: Aug-2010
Advisor: Byrne, Bernadette
Sponsor/Funder: Dstl
Author: Rana, Rohini R
Department: Molecular Biosciences
Publisher: Imperial College London
Qualification Level: Doctoral
Qualification Name: Doctor of Philosophy (PhD)
Appears in Collections:Molecular Biosciences PhD theses

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