Ligand Induced Conformational Changes within the Metal‐Dependent PI‐PLC from Streptomyces antibioticus

The objective of this study focused on understanding the conformational changes associated with calcium and inositol binding for the metal dependent phosphatidylinositol‐specific phospholipase C (PI‐PLC) from Streptomyces antibioticus ( hereafter referred to as SaPLC). By monitoring fluorescence quenching using both iodide and acrylamide—in the presence and absence of metal and/or inositol—we were able to deduce that one or more tryptophan residues became solvent exposed when either ligand was present. We then performed site directed mutagenesis studies and systematically changed each tryptophan residue to phenylalanine to understand which tryptophan amino acids are involved in the structural transition. These conformation changes were corroborated using 15 N‐HSQC NMR studies in an attempt to understand the global conformational changes associated with substrate and cofactor binding. Based on the recently solved x‐ray crystal structure completed by our lab, and this study, we propose that SaPLC undergoes significant active site rearrangement during substrate binding, which may help explain its novel stereochemical mechanism. Future studies include additional NMR experiments and fluorescent studies using terbium as an acceptor for the tryptophan fluorescence emission to better understand these active site conformational changes.