Spectroscopic characterization of the EF-hand domain of phospholipase C δ1:: Identification of a lipid interacting domain

The interaction of the isolated EF-hand domain of phospholipase C delta 1 with arachidonic acid (AA) was characterized using circular dichroism (CD) and fluorescence spectroscopy. The far-UV CD spectral changes indicate that AA binds to the EF domain. The near-UV CD spectra suggest that the orientations of aromatic residues in the pepticle are affected when AA binds to the protein. The fluorescence of the single intrinsic tryptophan located in EF I was enhanced by the addition of dodecylmaltoside (DDM) and AA suggesting that this region of the protein is involved in hydrophobic interactions. In the presence of a low concentration of DDM it was found that AA induced a change in fluorescence resonance energy transfer, which is indicative of a conformational change. The lipid induced conformational change may play a role in calcium binding because the isolated EF-hand domain did not bind Ca2+ in the absence of lipids, but Ca2+-dependent changes in the intrinsic tryptophan emission were observed when free fatty acids were present. These studies identify specific EF-hand domains as allosteric regulatory domains that require hydrophobic ligands such as lipids. (c) 2005 Elsevier Inc. All rights reserved.