| Abstract
| - C2 domains are protein modules found in numerous eukaryotic signaling proteins, where theirfunction is to target the protein to cell membranes in response to a Ca2+ signal. Currently, the structureof the interface formed between the protein and the phospholipid bilayer is inaccessible to high-resolutionstructure determination, but EPR site-directed spin-labeling can provide a detailed medium-resolutionview of this interface. To apply this approach to the C2 domain of cytosolic phospholipase A2 (cPLA2),single cysteines were introduced at all 27 positions in the three Ca2+-binding loops and labeled with amethanethiosulfonate spin-label. Altogether, 24 of the 27 spin-labeled domains retained Ca2+-activatedphospholipid binding. EPR spectra of these 24 labeled domains obtained in the presence and absence ofCa2+ indicate that Ca2+ binding triggers subtle changes in the dynamics of two localized regions withinthe Ca2+-binding loops: one face of the loop 1 helix and the junction between loops 1 and 2. However,no significant changes in loop structure were detected upon Ca2+ binding, nor upon Ca2+-triggered dockingto membranes. EPR depth parameters measured in the membrane-docked state allow determination of thepenetration depth of each residue with respect to the membrane surface. Analysis of these depth parameters,using an improved, generalizable geometric approach, provides the most accurate picture of penetrationdepth and angular orientation currently available for a membrane-docked peripheral protein. Finally, theobservation that Ca2+ binding does not trigger large rearrangements of the membrane-docking loops favorsthe electrostatic switch model for Ca2+ activation and disfavors, or places strong constraints on, theconformational switch model.
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