Biophysical characterization of the catalytic domain of guanine nucleotide exchange factor BopE from Burkholderia pseudomallei

Upadhyay, Abhishek and Williams, Christopher and Gill, Andrew C. and Philippe, Didier L. and Davis, Kenneth and Taylor, Lowrie A. and Stevens, Mark P. and Galyov, Edouard E. and Bagby, Stefan (2004) Biophysical characterization of the catalytic domain of guanine nucleotide exchange factor BopE from Burkholderia pseudomallei. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 1698 (1). pp. 111-119. ISSN 1570-9639

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Abstract

BopE is a type III secreted protein from Burkholderia pseudomallei, the aetiological agent of melioidosis. Like its Salmonella homologues SopE and SopE2, BopE is a guanine nucleotide exchange factor for Rho GTPases. It is thought that, in order to be secreted by the type III system, proteins must be unfolded or only partially folded. As part of a study of B. pseudomallei virulence proteins, we have expressed, purified and characterized the catalytic domain of BopE (amino acids 78-261). Analytical ultracentrifugation experiments in conjunction with analytical size exclusion chromatography show that BopE(78-261) is monomeric in aqueous solution. CD spectroscopy indicates that the protein is predominantly alpha-belical, with predicted secondary structure composition of 59% alpha-helix and 7% beta-strand. NMR spectroscopy confirms that BopE(78-261) adopts a single, stable conformation. In differential scanning calorimetry experiments, thermal denaturation of BopE(78-261) (T-m 52 degreesC) is reversible. Also, the secondary structure composition of BopE78-261 changes little over a range of pH values from 3.5 to 10.5. BopE may therefore fold spontaneously to a functional form upon secretion into the host cell cytoplasm, and retains a native or native-like fold in varied environments. These properties are likely to be advantageous for a secreted bacterial effector protein.

Keywords:BopE, Burkholderia pseudomallei, circular dichroism, differential scanning calorimetry, guanine nucleotide exchange factor, Type III secretion system
Subjects:C Biological Sciences > C760 Biomolecular Science
Divisions:College of Science
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ID Code:29567
Deposited On:16 Mar 2018 15:01

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