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

Minae Kobayashi, Zygmunt Gryczynski, Joanna Lukomska, Jianwen Feng, Mary F. Roberts, Joseph R. Lakowicz, Jon W. Lomasney

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

The interaction of the isolated EF-hand domain of phospholipase C δ1 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 peptide are affected when AA binds to the protein. The fluorescence of the single intrinsic tryptophan located in EF1 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.

Original languageEnglish
Pages (from-to)191-203
Number of pages13
JournalArchives of Biochemistry and Biophysics
Volume440
Issue number2
DOIs
StatePublished - 15 Aug 2005

Fingerprint

EF Hand Motifs
Type C Phospholipases
Arachidonic Acid
Lipids
Circular Dichroism
Dichroism
Tryptophan
Circular dichroism spectroscopy
Fluorescence Resonance Energy Transfer
Fluorescence Spectrometry
Fluorescence spectroscopy
Hydrophobic and Hydrophilic Interactions
Nonesterified Fatty Acids
Proteins
Fluorescence
Ligands
Calcium
Peptides

Keywords

  • Allosterism
  • Arachidonic acid
  • Calcium binding
  • Circular dichroism
  • EF-hand domain
  • FRET
  • Fatty acids
  • Fluorescence spectroscopy
  • Hydrophobic interaction
  • Phospholipase C

Cite this

Kobayashi, Minae ; Gryczynski, Zygmunt ; Lukomska, Joanna ; Feng, Jianwen ; Roberts, Mary F. ; Lakowicz, Joseph R. ; Lomasney, Jon W. / Spectroscopic characterization of the EF-hand domain of phospholipase C δ1 : Identification of a lipid interacting domain. In: Archives of Biochemistry and Biophysics. 2005 ; Vol. 440, No. 2. pp. 191-203.
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Spectroscopic characterization of the EF-hand domain of phospholipase C δ1 : Identification of a lipid interacting domain. / Kobayashi, Minae; Gryczynski, Zygmunt; Lukomska, Joanna; Feng, Jianwen; Roberts, Mary F.; Lakowicz, Joseph R.; Lomasney, Jon W.

In: Archives of Biochemistry and Biophysics, Vol. 440, No. 2, 15.08.2005, p. 191-203.

Research output: Contribution to journalArticle

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