22 research outputs found
Studies on phosphatidylserine by tandem quadrupole and multiple stage quadrupole ion-trap mass spectrometry with electrospray ionization: Structural characterization and the fragmentation processes
Binding of the PX domain of p47phox to phosphatidylinositol 3,4-bisphosphate and phosphatidic acid is masked by an intramolecular interaction
INFLUENCE OF AGGREGATE COATING ON RELAXATIONS IN THE SYSTEMS OF IRON OXIDE NANOPARTICLES
Calcium Binding Rigidifies the C2 Domain and the Intradomain Interaction of GIVA Phospholipase A2 as Revealed by Hydrogen/Deuterium Exchange Mass Spectrometry*
The GIVA phospholipase A2 (PLA2) contains two
domains: a calcium-binding domain (C2) and a catalytic domain. These domains
are linked via a flexible tether. GIVA PLA2 activity is
Ca2+-dependent in that calcium binding promotes protein docking to
the phospholipid membrane. In addition, the catalytic domain has a lid that
covers the active site, presumably regulating GIVA PLA2 activity.
We now present studies that explore the dynamics and conformational changes of
this enzyme in solution utilizing peptide amide hydrogen/deuterium (H/D)
exchange coupled with liquid chromatographymass spectrometry (DXMS) to probe
the solvent accessibility and backbone flexibility of the C2 domain, the
catalytic domain, and the intact GIVA PLA2. We also analyzed the
changes in H/D exchange of the intact GIVA PLA2 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” region between the C2 and
catalytic domains. The slow exchanging region of the C2 domain is in tight
proximity to the catalytic domain. 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 in GIVA PLA2 activation
X-Ray Reflectivity Studies of cPLA(2)α-C2 Domains Adsorbed onto Langmuir Monolayers of SOPC
X-ray reflectivity is used to study the interaction of C2 domains of cytosolic phospholipase A(2) (cPLA(2)α-C2) with a Langmuir monolayer of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) supported on a buffered aqueous solution containing Ca(2+). The reflectivity is analyzed in terms of the known crystallographic structure of cPLA(2)α-C2 domains and a slab model representing the lipid layer to yield an electron density profile of the lipid layer and bound C2 domains. This new method of analysis determines the angular orientation and penetration depth of the cPLA(2)α-C2 domains bound to the SOPC monolayer, information not available from the standard slab model analysis of x-ray reflectivity. The best-fit orientation places the protein-bound Ca(2+) ions within 1 Å of the lipid phosphate group (with an accuracy of ±3 Å). Hydrophobic residues of the calcium-binding loops CBL1 and CBL3 penetrate deepest into the lipid layer, with a 2 Å penetration into the tailgroup region. X-ray measurements with and without the C2 domain indicate that there is a loss of electrons in the headgroup region of the lipid monolayer upon binding of the domains. We suggest that this is due to a loss of water molecules bound to the headgroup. Control experiments with a non-calcium buffer and with domain mutants confirm that the cPLA(2)α-C2 binding to the SOPC monolayer is Ca(2+)-dependent and that the hydrophobic residues in the calcium-binding loops are critical for membrane binding. These results indicate that an entropic component (due to water loss) as well as electrostatic and hydrophobic interactions contributes to the binding mechanism