Probing the Ca²⁺ Switch of the Neuronal Ca²⁺ 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.

Turning On Fluorescence with Thiols – Synthetic and Computational Studies on Diaminoterephthalates and Monitoring the Switch of the Ca2+ Sensor Recoverin

“…[13] Results and Discussion Besides the labeling of recoverin, which we report herein, we are therefore aiming to use NiWa dyes for other applications in the future, e.g.…”

“…NH protons are not detectable 13. C{ 1 H} NMR (125 MHz, CDCl 3 , 330 K): δ = 20.38 (CH 3 ), 35.72 (CH 2 ), 37.23 (CH 2 ), 38.47 (CH 2 ), 41.11 (CH 2 ), 42.02 (CH 2 ), 51.91 (2 CH 3 ), 67.50 (CH), 114.46 (CH), 114.60 (CH), 117.46 (C), 117.74 (C), 134.16 (2 CH), 140.50 (C), 140.93 (C), 168.02 (C), 168.11 (C), 169.64 (C), 170.54 (2 C), 173.05 (C), 173.31 (C) ppm.…”

Protein and Signaling Networks in Vertebrate Photoreceptor Cells

“…However, the protein dynamics of Ca 2+ -triggered conformational changes show significant differences between mammalian GCAP1 and GCAP2 on a nanosecond time scale. Fluorescence lifetime and anisotropy measurements of GCAP1 and GCAP2 that were site specifically labeled with the dye Alexa647 revealed different movements of secondary structural elements (Kollmann et al, 2012 ; Robin et al, 2015 ). In the case of GCAP1 these findings were further supported by molecular dynamics simulation showing that GCAP1 undergoes a twisted accordion-like movement upon changing Ca 2+ -concentration (Robin et al, 2015 ).…”

“…In the case of GCAP1 these findings were further supported by molecular dynamics simulation showing that GCAP1 undergoes a twisted accordion-like movement upon changing Ca 2+ -concentration (Robin et al, 2015 ). GCAP2 in contrast responds to a change in Ca 2+ by an up-and-down or piston-like movement of an α-helix between amino acid positions 111 and 131 (Kollmann et al, 2012 ; Figure 5 ). A main conclusion from these studies was that two structurally similar proteins can differ significantly in their conformational structural dynamics thereby providing structural framework for their different regulatory responses.…”

The guanylate cyclase signaling system in zebrafish photoreceptors

“…In addition, the affinity of the GCAP1–ROS–GC1 interaction depends on the myristoyl modification [54–56]. In contrast, bovine GCAP2 shows an almost identical activation pattern in its myristoylated or non‐myristoylated form, however, the myristoyl group in GCAP2 seems to be more flexible and is involved in stabilizing the Ca 2+ ‐bound conformation [57,58]. These findings raised the following questions, (a) whether zGCAPs are expressed in myristoylated or non‐myristoylated forms; (b) and if so, whether these forms differ in their key properties.…”