X-ray Reflectivity Studies of cPLA?-C2 Domains Adsorbed onto Langmuir Monolayers of SOPC
X-ray reflectivity is used to study the interaction of C2 domains of cytosolic phospholipase A2 (cPLA{sub 2{alpha}}-C2) with a Langmuir monolayer of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) supported on a buffered aqueous solution containing Ca{sup 2+}. The reflectivity is analyzed in terms of the known crystallographic structure of cPLA2{alpha}-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{sub 2{alpha}}-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{sup 2+} ions within 1 Angstrom of the lipid phosphate group (with an accuracy of {+-}3 Angstroms). Hydrophobic residues of the calcium-binding loops CBL1 and CBL3 penetrate deepest into the lipid layer, with a 2 Angstrom 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{sub 2{alpha}}-C2 binding to the SOPC monolayer is Ca{sup 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.
- Research Organization:
- Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
- Sponsoring Organization:
- Doe - Office Of Science
- DOE Contract Number:
- DE-AC02-98CH10886
- OSTI ID:
- 913831
- Report Number(s):
- BNL-78399-2007-JA; BIOJAU; TRN: US200804%%93
- Country of Publication:
- United States
- Language:
- English
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