Structural characterization of metalloprotease vibriolysin of cholera pathogen Vibrio cholerae.
Vibriolysin is among several zinc metalloproteases produced by Vibrio cholerae. It is involved in the molecular pathogenicity of cholera. Here, we cloned and expressed full-length vibriolysin gene from V. cholerae. Electrophoretic and mass spectrometric data showed that the N-terminal pro-peptide was removed from pro-vibriolysin generating a 45-kDa segment containing the metalloprotease plus the C-terminal domains, and the 35 kDa metalloprotease. The 35 kDa metalloprotease segment of vibriolysin was purified to homogeneity using ion-exchange and gel filtration chromatography. Circular dichroism (CD) analysis of vibriolysin indicated α+β secondary structure, similar to other closely related metalloproteases of known structure. Positive dichroic absorption maxima in near-UV CD spectrum provided evidence for bound metal atom(s). Dynamic Light Scattering (DLS) measurements at different pHs were also performed to establish the aggregational properties of purified vibriolysin in solution. The results of DLS studies revealed that vibriolysin exists as a homomer with a hydrodynamic radius of 56.7 nm ± 2% under physiological conditions and remains catalytic when BSA was used as a protein substrate. While, extreme acidic (pH 3.5-5.4; R(H) = 65-239 nm) and alkaline (pH 8.5-9.4; R(H) = 57-72 nm) buffering conditions induced further aggregation of vibriolysin, without any trace of the monomeric state in solution.
International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan.
Asif
A
Iqbal
M Kamran
MK
Azim
Naghma
N
Hashmi
S Abid
SA
Ali
S Ghulam
SG
Musharaf
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002771:Cholera / Q000382:microbiology*; D008027:Light; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012542:Scattering, Radiation; D014734:Vibrio cholerae / Q000201:enzymology*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C073831:vibriolysin protein, Vibrio proteolyticus