title: Probing the Ca(2+) switch of the neuronal Ca(2+) sensor GCAP2 by time-resolved fluorescence spectroscopy.
abstract: We report fluorescence lifetime and rotational anisotropy measurements of the fluorescent dye Alexa647 attached to the guanylate cyclase-activating protein 2 (GCAP2), an intracellular myristoylated calcium sensor protein operating in photoreceptor cells. By linking the dye to different protein regions critical for monitoring calcium-induced conformational changes, we could measure fluorescence lifetimes and rotational correlation times as a function of myristoylation, calcium, and position of the attached dye, while GCAP2 was still able to regulate guanylate cyclase in a Ca(2+)-sensitive manner. We observe distinct site-specific variations in the fluorescence dynamics when externally changing the protein conformation. A clear reduction in fluorescence lifetime suggests that in the calcium-free state a dye marker in amino acid position 131 senses a more hydrophobic protein environment than in position 111. Saturating GCAP2 with calcium increases the fluorescence lifetime and hence leads to larger exposure of position 111 to the solvent and at the same time to a movement of position 131 into a hydrophobic protein cleft. In addition, we find distinct, biexponential anisotropy decays reflecting the reorientational motion of the fluorophore dipole and the dye/protein complex, respectively. Our experimental data are well described by a "wobbling-in-a-cone" model and reveal that for dye markers in position 111 of the GCAP2 protein both addition of calcium and myristoylation results in a pronounced increase in orientational flexibility of the fluorophore. Our results provide evidence that the up-and-down movement of an α-helix that is situated between position 111 and 131 is a key feature of the dynamics of the protein-dye complex. Operation of this piston-like movement is triggered by the intracellular messenger calcium.
authors.affiliation: Ultrafast Nano-Optics, Institute of Physics, University of Oldenburg, D-26111 Oldenburg, Germany.
authors.forename: Heiko
authors.initials: H
authors.lastname: Kollmann
authors.forename: Simon F
authors.initials: SF
authors.lastname: Becker
authors.forename: Javid
authors.initials: J
authors.lastname: Shirdel
authors.forename: Alexander
authors.initials: A
authors.lastname: Scholten
authors.forename: Anna
authors.initials: A
authors.lastname: Ostendorp
authors.forename: Christoph
authors.initials: C
authors.lastname: Lienau
authors.forename: Karl-Wilhelm
authors.initials: KW
authors.lastname: Koch
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002232:Carbocyanines / Q000032:analysis*; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics; D005454:Fluorescence Polarization / Q000379:methods; D005456:Fluorescent Dyes / Q000032:analysis*; D015870:Gene Expression; D051604:Guanylate Cyclase-Activating Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D010786:Photoreceptor Cells / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: C569686:Alexa Fluor 647; D002232:Carbocyanines; D005456:Fluorescent Dyes; D051604:Guanylate Cyclase-Activating Proteins; D002118:Calcium