Mutations on N-terminal region of Taiwan cobra phospholipase A2 result in structurally distorted effects

✍ Scribed by Yi-Ling Chiou; Shinne-Ren Lin; Long-Sen Chang

• Publisher: John Wiley and Sons
• Year: 2008
• Tongue: English
• Weight: 315 KB
• Volume: 14
• Category: Article
• ISSN: 1075-2617
• DOI: 10.1002/psc.1020

✦ Synopsis

Abstract

In the present study, three Taiwan cobra PLA~2~ variants were prepared by adding an extra N‐terminal Met, substituting Asn‐1 by Met or deleting the N‐terminal heptapeptide. Recombinant PLA~2~ mutants were expressed in Escherichia coli (E. coli), and purified to homogeneity by reverse phase HPLC. Fluorescence measurement showed that the hydrophobic character of the catalytic site, the microenvironment of Trp residues and energy transfer from excited Trp to 8‐anilinonaphthalene sulfonate (ANS) were affected by N‐terminal mutations. An alteration in the structural flexibility of the active site was noted with the mutants lacking the N‐terminal heptapeptide or with an extra N‐terminal Met added as evidenced by the inability of the two variants to bind with Ba^2+^. Moreover, modification of Lys residues and energy transfer within the protein‐ANS complex revealed that the Ca^2+^‐induced change in the global structure of PLA~2~ was different from that in N‐terminal variants. Together with the fact that an ‘activation network’ connects the N‐terminus with the active site, our data suggest that mutagenesis on the N‐terminal region affects directly the fine structure of the catalytic site, which subsequently transmits its influence in altering the structure outside the active site of PLA~2~. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.