Petra Popovics 1 , Jin Lu 2 , L. Nadia Kamil 1 , Kevin Morgan 3 , Robert P. Millar 3 ' 4 , Ralf Schmid 5 , Claudia A. Blindauer 2 and Alan J. Stewart 1 * 1 School of Medicine, University of St Andrews, St Andrews, UK. 2 Department of Chemistry, University of Warwick, Coventry, UK. 3 MRC Human Reproductive Sciences Unit, Queen’s Medical Research Institute, Edinburgh, UK. 4 Mammal Research Institute, University of Pretoria, Pretoria, South Africa. 5 Department of Biochemistry, University of Leicester, Leicester, UK. 'Correspondence: Dr Alan J. Stewart, Medical and Biological Sciences Building, University of St Andrews, North Haugh, St Andrews, Fife, KY16 9TF, UK. Tel: +44 1334 463546; Fax: +44 1334 463482; E-mail: ajs21@st- andrews.ac.uk. Abbreviations: [Ca 2+ ] i , intracellular free Ca 2+ concentration; CaM, calmodulin; DAG, 1,2- diacylglycerol; DMEM, Dulbecco’s modified Eagle’s medium; IP 3 , inositol 1,4,5-trisphosphate; NCS-1, neuronal calcium sensor-1; NOESY, nuclear Overhauser effect spectroscopy; PH, pleckstrin homology; PIP 2 , phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; TOCSY, total correlation spectroscopy. Abstract Phospholipase C-ɳ(PLCɳ) enzymes are a class of phosphatidylinositol 4,5-bisphosphate- hydrolyzing enzymes involved in intracellular signaling. PLCɳ2 can sense Ca 2+ (stimulated by ~1 µM free Ca 2+ ) suggesting that it can amplify transient Ca 2+ signals. PLCɳ enzymes possess an EF-hand domain composed of two EF-loops; a canonical 12-residue loop (EF-loop 1) and a non-canonical 13-residue loop (EF-loop 2). Ca 2+ -binding to synthetic peptides corresponding to EF-loops 1 and 2 of PLCɳ2 and EF-loop 1 of calmodulin (as a control) was examined by 2D-[ 1 H, 1 H] TOCSY NMR. Both PLCɳ2 EF-loop peptides bound Ca 2+ in a similar manner to that of the canonical calmodulin EF-loop 1, particularly at their N-terminus. A molecular model of the PLCɳ2 EF-hand domain, constructed based upon the structure of calmodulin,