Biophysical characterization of the catalytic domain of guanine nucleotide exchange factor BopE from Burkholderia pseudomallei.
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-helical, 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 degrees C) is reversible. Also, the secondary structure composition of BopE(78-261) 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.
Upadhyay
Abhishek
A
Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK.
Williams
Christopher
C
Gill
Andrew C
AC
Philippe
Didier L
DL
Davis
Kenneth
K
Taylor
Lowrie A
LA
Stevens
Mark P
MP
Galyov
Edouard E
EE
Bagby
Stefan
S
D000595:Amino Acid Sequence; D016957:Burkholderia pseudomallei / Q000235:genetics / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D020134:Catalytic Domain; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D012475:Salmonella / Q000235:genetics; D016415:Sequence Alignment
D020662:Guanine Nucleotide Exchange Factors