Changes in the Secondary and Tertiary Structures of Secreted Phospholipase A2 upon Activation

Abstract

Activation of human pancreatic phospholipase A2 (PLA2) in the presence of DPPC/DPPG (7:3) vesicles was induced by a temperature shift from 4 to 38 oC. PLA2 activity was monitored by changes in fluorescence of bis-Pyrene-PC (2.5 mol % in the membranes), while simultaneous far- and near-UV circular dichroism (CD) spectra identified changes in the secondary and tertiary structures of the protein in real time. The 4-to-38 oC temperature shift caused dramatic changes in both bis-Pyrene-PC fluorescence and the protein CD spectra. The monomer fluorescence signal of bis-Pyrene-PC rapidly increased and the excimer signal decreased, demonstrating PLA2 activation. Drastic weakening in the α-helical CD signal of the protein, i.e., a 20% decrease in the n-π* transition intensity at 222 nm, was detected upon enzyme activation. The α-helical signal exhibited a significantly smaller change upon a similar temperature shift under non-catalytic conditions (1 mM EGTA), while little changes were detected in the absence of lipid. Strong changes in the tertiary structure during PLA2 activation were also identified. Initially, at 4 oC, the near-UV CD spectra showed a weak negative band around 280 nm. Upon a shift to 38 oC, strong positive CD bands rapidly developed around 250 and 280 nm, implying significant changes in the conformation and/or the microenvironment of Tyr and Trp side chains of PLA2, possibly accompanied with a global tertiary structure perturbation associated with deformation of the abundant disulfide bonds in the protein. These experiments provide new information on the structure-function relationship of PLA2 by near-simultaneous measurements of PLA2 activity and its secondary and tertiary structures.