C2 domains are protein modules found in numerous eukaryotic signaling proteins, where their function is to target the protein to cell membranes in response to a Ca 2+ signal. Currently, the structure of the interface formed between the protein and the phospholipid bilayer is inaccessible to high-resolution structure determination, but EPR site-directed spin-labeling can provide a detailed medium-resolution view of this interface. To apply this approach to the C2 domain of cytosolic phospholipase A 2 (cPLA 2 ), single cysteines were introduced at all 27 positions in the three Ca 2+ -binding loops and labeled with a methanethiosulfonate spin-label. Altogether, 24 of the 27 spin-labeled domains retained Ca 2+ -activated phospholipid binding. EPR spectra of these 24 labeled domains obtained in the presence and absence of Ca 2+ indicate that Ca 2+ binding triggers subtle changes in the dynamics of two localized regions within the Ca 2+ -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 Ca 2+ binding, nor upon Ca 2+ -triggered docking to membranes. EPR depth parameters measured in the membrane-docked state allow determination of the penetration 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 penetration depth and angular orientation currently available for a membrane-docked peripheral protein. Finally, the observation that Ca 2+ binding does not trigger large rearrangements of the membrane-docking loops favors the electrostatic switch model for Ca 2+ activation and disfavors, or places strong constraints on, the conformational switch model. Cellular signals are transmitted by a variety of mechanisms, including the release of small molecule second messengers such as Ca 2+ or phosphoinositides, protein translocation between cellular compartments, and protein posttranslational modification. C2 domains are protein-signaling modules found in numerous signaling proteins (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). Usually, the function of such C2 domains is to trigger the translocation of proteins to specific cellular membranes in response to a Ca 2+ signal. This targeting increases the likelihood of interaction of the signaling protein with its downstream target, often a membrane-bound lipid or protein.Cytosolic phospholipase A 2 (cPLA 2 ) 1 hydrolyzes lipids containing arachadonic acid and thereby releases this important precursor from nuclear and endoplasmic reticulum (ER) † Support provided by NIH Grant GM R01-63235 (to J.J.F.).© 2003 American Chemical Society * To whom correspondence should be addressed. falke@colorado.edu. Tel: (303) 492-3503. 1 Abbreviations: cPLA 2 , cytosolic phospholipase A 2 ; PS, phosphati-dylserine; PC, phosphatidylcholine; PE, phosphoethanolamine; MTSSL, 1-oxyl-2,2,5,5-tetramethyl-Δ 3 -pyrroline-3-methyl methanethiosulfonate; NiEDDA, Ni(II) ethylenediamine...