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

doi:10.1016/j.abb.2005.06.014

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^*

^*Corresponding author for this work

Pathology

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12 Scopus citations

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 language	English (US)
Pages (from-to)	191-203
Number of pages	13
Journal	Archives of biochemistry and biophysics
Volume	440
Issue number	2
DOIs	https://doi.org/10.1016/j.abb.2005.06.014
State	Published - Aug 15 2005

Funding

This work was supported by National Institutes of Health Grants HL55591 and HL03961 to J.W.L, and GM60418 to M.F.R. We thank Dr. Carl Waltenbaugh for letting us use a fluorescence plate reader for preliminary results and Dr. John Solaro and Dr. Tomoyoshi Kobayashi at UIC for letting us use their instrument. We also thank Ms. Li Lin for helping us make some of the constructs. For anisotropy measurement we thankfully acknowledge the use of instruments in the Keck Biophysics Facility at Northwestern University [ http://www.biochem.northwestern.edu/Keck/keckmain.html ].

Keywords

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

ASJC Scopus subject areas

Molecular Biology
Biophysics
Biochemistry

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10.1016/j.abb.2005.06.014

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@article{dc7da2542fc54f25bec5a24985429b74,

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

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.",

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

author = "Minae Kobayashi and Zygmunt Gryczynski and Joanna Lukomska and Jianwen Feng and Roberts, {Mary F.} and Lakowicz, {Joseph R.} and Lomasney, {Jon W.}",

note = "Funding Information: This work was supported by National Institutes of Health Grants HL55591 and HL03961 to J.W.L, and GM60418 to M.F.R. We thank Dr. Carl Waltenbaugh for letting us use a fluorescence plate reader for preliminary results and Dr. John Solaro and Dr. Tomoyoshi Kobayashi at UIC for letting us use their instrument. We also thank Ms. Li Lin for helping us make some of the constructs. For anisotropy measurement we thankfully acknowledge the use of instruments in the Keck Biophysics Facility at Northwestern University [ http://www.biochem.northwestern.edu/Keck/keckmain.html ].",

year = "2005",

month = aug,

day = "15",

doi = "10.1016/j.abb.2005.06.014",

language = "English (US)",

volume = "440",

pages = "191--203",

journal = "Archives of biochemistry and biophysics",

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T1 - Spectroscopic characterization of the EF-hand domain of phospholipase C δ1

T2 - Identification of a lipid interacting domain

AU - Kobayashi, Minae

AU - Gryczynski, Zygmunt

AU - Lukomska, Joanna

AU - Feng, Jianwen

AU - Roberts, Mary F.

AU - Lakowicz, Joseph R.

AU - Lomasney, Jon W.

N1 - Funding Information: This work was supported by National Institutes of Health Grants HL55591 and HL03961 to J.W.L, and GM60418 to M.F.R. We thank Dr. Carl Waltenbaugh for letting us use a fluorescence plate reader for preliminary results and Dr. John Solaro and Dr. Tomoyoshi Kobayashi at UIC for letting us use their instrument. We also thank Ms. Li Lin for helping us make some of the constructs. For anisotropy measurement we thankfully acknowledge the use of instruments in the Keck Biophysics Facility at Northwestern University [ http://www.biochem.northwestern.edu/Keck/keckmain.html ].

PY - 2005/8/15

Y1 - 2005/8/15

N2 - 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.

AB - 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.

KW - Allosterism

KW - Arachidonic acid

KW - Calcium binding

KW - Circular dichroism

KW - EF-hand domain

KW - FRET

KW - Fatty acids

KW - Fluorescence spectroscopy

KW - Hydrophobic interaction

KW - Phospholipase C

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U2 - 10.1016/j.abb.2005.06.014

DO - 10.1016/j.abb.2005.06.014

M3 - Article

C2 - 16054586

AN - SCOPUS:23744471645

SN - 0003-9861

VL - 440

SP - 191

EP - 203

JO - Archives of biochemistry and biophysics

JF - Archives of biochemistry and biophysics

IS - 2

ER -

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

Abstract

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Keywords

ASJC Scopus subject areas

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