Premium
Calcium binding effects on GIVA Phospholipase A2 mapped by Hydrogen/Deuterium exchange mass spectrometry
Author(s) -
Hsu YuanHao,
Burke John E,
Woods Virgil E.,
Dennis Edward A.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.611.19
Subject(s) - chemistry , hydrogen–deuterium exchange , calcium , mass spectrometry , c2 domain , deuterium , membrane , biophysics , catalysis , binding domain , binding site , phospholipase , enzyme , crystallography , biochemistry , chromatography , organic chemistry , physics , quantum mechanics , biology
The GIVA phospholipase A2 (PLA2) contains two domains: a calcium binding domain (C2) and a catalytic domain. GIVA PLA2 activity is Ca2+ dependent in that calcium binding promotes protein docking to the phospholipid membrane. The C2 domain and possibly the catalytic domain penetrate the lipid surface. We now present studies that explore the dynamics and conformational changes of this enzyme in solution utilizing peptide amide hydrogen/deuterium exchange coupled with liquid chromatography‐mass spectrometry (DXMS) to probe the solvent accessibility and backbone flexibility of the GIVA PLA2. We also analyzed the changes in H/D exchange of the GIVA PLA2 holoenzyme upon Ca2+ binding. The DXMS results showed a fast H/D exchanging lid and a slow exchanging central core. The C2 domain showed two distinct regions: a fast exchanging region facing away from the catalytic domain and a slow exchanging region present in the cleft between the C2 and catalytic domains. The effects of Ca2+ binding on GIVA PLA2 are localized in the C2 domain and suggest that binding of two distinct Ca2+ ions causes tightening up of the regions that surround the anion hole at the tip of the C2 domain. This conformational change may be the initial step for membrane penetration.