Insight into the conformational dynamics of specific regions of porcine pancreatic phospholipase A2 from a time-resolved fluorescence study of a genetically inserted single tryptophan residue
The effects of Ca2+ and substrate analogue binding on the conformational dynamics of porcine pancreas phospholipase A2 (PLA2) in different regions was explored by combining site-directed mutagenesis and time-resolved fluorescence measurements. The single tryptophan residue (Trp-3) of the wild-type protein (W3), in the α-helix A, was replaced by a phenylalanine residue (W3F), whereafter Trp was substituted either for leucine-31 (W31), located in the calcium binding loop, or for phenylalanine-94 (W94), located at the "back side" of the enzyme. Furthermore, mutants lacking the 62-66 sequence were constructed with the Trp at position 3 (ΔW3) or 31 (ΔW31). The total fluorescence intensity decays of Trp in each protein, in the protein-calcium and the protein-calcium-substrate analogue complexes, analyzed by the maximum entropy method (MEM) can be interpreted as distributions of separated lifetime classes. In the case of the W94 mutant, a major short-lived excited-state population (τ ~ 50 ps) is observed, probably deactivated by the interaction with two proximate disulfide bridges via a radiationless process. For the four other mutants, the respective barycenters of the four lifetime classes display comparable values, but the amplitude distributions are different for Trp-3 and Trp-31. The rotational mobility of the Trp residue varies along the peptide chain. Trp-3 experiences only a fast hindered motion. Trp-31 is sensitive to an additional local flexibility that is absent in the N-terminal part of the protein. The largest wobbling angle is observed at position 94. No effect of calcium binding occurs on the lifetime distribution of the Trp-3 and Trp-94 residues. Their mobilities are not affected. In contrast, calcium binding displays a strong influence on the excited-state population distribution of Trp-31. A major population decaying with the longest lifetime is selected in the W31 protein and contributes to ~50% of the decay. The local flexibility and the amplitude of motion of Trp-31 is wider in the protein-calcium complex than in the unliganded protein. Binding of the monomeric substrate analogue n-dodecylphosphocholine (C12PN) in the presence of calcium slightly affects the Trp-3 excited-state population distribution and its mobility. Trp-31 is more sensitive to this binding. In particular, a more restricted rotation of the Trp-31 residue and a decrease of the peptide local flexibility as protein-calcium complexes are observed in both the W31 and ΔW31 mutants. The binding of the micellar substrate analogue n-hexadecylphosphocholine (C16PN) in the presence of calcium considerably modifies the excited-state population distribution of Trp-3 in the W3 and the ΔW3 proteins. One major broad lifetime population (centered at ~2.6 ns) is selected for the wild-type protein. The internal motion of Trp3 in both the wild-type and the ΔW3 proteins is strongly reduced. In contrast to these strong effects on position 3, the excited-state population distribution of Trp31 is only slightly affected by micelle binding as compared to monomer binding. No large changes in the mobility can be observed. The lifetime distribution of Trp-94 is not changed upon micelle binding, but the barycenter value of the major short lifetime is shifted to an even shorter value. The rotational motion is slower and its amplitude is higher in the protein-micelle complex. The results are discussed in terms of specific modulations of the protein structure and flexibility by calcium and substrate analogue binding