Mapping Calcium-Sensitive Regions in the Neuronal Calcium Sensor GCAP2 by Site-Specific Fluorescence Labeling.
Myristoylation of most neuronal calcium sensor proteins, a group of EF-hand calcium-binding proteins mainly expressed in neuronal tissue, can have a strong impact on protein dynamics and functional properties. Intracellular oscillations of the free Ca(2+) concentration can trigger conformational changes in Ca(2+) sensors. The position and possible movements of the myristoyl group in the photoreceptor cell-specific Ca(2+) sensor GCAP2 are not well-defined but appear to be different from those of the highly homologous cognate GCAP1. We designed and applied a new group of diaminoterephthalate-derived fluorescent probes to label GCAP2 at a covalently attached 12-azido-dodecanoic acid (a myristoyl substitute) and at cysteine residues in critical positions. Fluorescence emission of dye-labeled GCAP2 decreased when going from low (10(-9) M) to high [Ca(2+)] (10(-3) M), reaching a half-maximal effect of fluorescence emission at 0.44 ± 0.07 μM. The modified acyl group can therefore monitor changes in the protein conformation during binding and dissociation of Ca(2+) in the physiological range of free [Ca(2+)]. However, fluorescence quenching studies showed that the dye-acyl chain was shielded from the quencher by an adjacent polypeptide region. Further probing three cysteine positions (C35, C111, and C131) by dye labeling revealed that all positions were also sensitive to a change in [Ca(2+)], but only one (C131) was sensitive to a change in [Mg(2+)]. We suggest a scenario during illumination of the photoreceptor cell in which Ca(2+) dissociates first from low and medium affinity binding sites. These steps are sensed by dyes in cysteines at positions 35 and 111. Release of Ca(2+) from high affinity sites is sensed by regions adjacent to the dye-labeled fatty acid and involves the critical conformational change leading to activating guanylate cyclase.
Biochemistry Group, Department of Neurosciences, Carl von Ossietzky Universität Oldenburg , D-26111 Oldenburg, Germany.
Stefan
S
Sulmann
Institut für Chemie, Carl von Ossietzky Universität Oldenburg , D-26111 Oldenburg, Germany.
Melanie
M
Wallisch
Biochemistry Group, Department of Neurosciences, Carl von Ossietzky Universität Oldenburg , D-26111 Oldenburg, Germany.
Alexander
A
Scholten
Institut für Chemie, Carl von Ossietzky Universität Oldenburg , D-26111 Oldenburg, Germany.
Jens
J
Christoffers
Biochemistry Group, Department of Neurosciences, Carl von Ossietzky Universität Oldenburg , D-26111 Oldenburg, Germany.
Karl-Wilhelm
KW
Koch
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D002417:Cattle; D005456:Fluorescent Dyes / Q000737:chemistry*; D051604:Guanylate Cyclase-Activating Proteins / Q000737:chemistry*; D011485:Protein Binding; D013194:Staining and Labeling
D005456:Fluorescent Dyes; D051604:Guanylate Cyclase-Activating Proteins; D002118:Calcium