Schematic representation of domain structure and genomic organization of Epac protein isoforms. Epac1 and Epac2 consist of the N-terminal regulatory region and the C-terminal catalytic region in common, which are composed of functional multi-domains. The regulatory region contains a cyclic nucleotide-binding (CNB) domain and a dishevelled, Egl-10, Pleckstrin (DEP) domain. The CNB domain of Epac1 and CNB-B domain of Epac2 bind cAMP with a high affinity leading to Epac protein activation. The extra CNB-A domain of Epac2A1 and Epac2A2 bind cAMP with a relatively low affinity compared with the conserved CNB-B domain and is not involved in activation of Epac2. The Dishevelled, Egl-10, Pleckstrin (DEP) domain has a role in the subcellular localization of Epac protein. In the catalytic region, a RAS exchange motif (REM) domain interacting with the guanine nucleotide exchange factor (GEF) region stabilizes a GEF for Ras-like small GTPases (RasGEF) domain which is responsible for biological function of Epac protein. The RAS-association (RA) domain regulates perinuclear localization of Epac1 and plasma membrane localization of Epac2.
(A) In fluorescence correlation spectroscopy, a confocal laser illumination spot is used to monitor the intensity fluctuation due to fluorescence molecules diffusing in and out of the focus. The auto-correlation function (ACF) of the resulting time trace is used to compute the particle number (N, from the y-intercept) and residence time (τd, from the half-point of the ACF decay) of the fluorescent molecule in the laser focus, which is related to the diffusion coefficient by w2 = 4Dτd. In this example, a GFP-labeled protein on a supported lipid bilayer is excited by a blue (488 nm) laser. (B) In total internal relfection fluorescence microscopy (TIRFM) single molecule tracking, the displacement of the particle between each frame are reconstructed into particle trajectories (example trajectory shown in inset). The step size distribution may be used to calculate the diffusion coefficient and the relative population of multiple diffusing species, following the step size equation P(r) (Equations 5 and 6 for single and two species, respectively). The fit to a single species step size distribution is shown. (C) Left: theoretical binding curves for a dimerization reaction for a wide range of dissociation constants, right: simulated FCS data using experimentally determined diffusion coefficients for monomers and dimers for the same dissociation constants.
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