Shaping
the temporal response of photoreceptors is facilitated
by a well-balanced second messenger cascade, in which two neuronal
Ca<sup>2+</sup>-sensor proteins operate in a sequential relay mechanism.
Although they share structurally similar sensing units, they differentially
activate the same target protein. Here, as a prototypical case in
Ca<sup>2+</sup>-mediated signal processing, we investigate differential
cellular responsiveness in protein conformational dynamics on a nanosecond
time scale. For this, we have site-specifically labeled cysteine residues
in guanylate cyclase-activating protein GCAP1 by the fluorescent dye
Alexa647 and probed its local environment via time-resolved fluorescence
spectroscopy. Fluorescence lifetime and rotational anisotropy measurements
reveal a distinct structural movement of the polypeptide chain around
position 106 upon release of Ca<sup>2+</sup>. This is supported by
analyzing the diffusional dye motion in a wobbling-in-a-cone model
and by molecular dynamics simulations. We conclude that GCAP1 and
its cellular cognate GCAP2 operate by distinctly different switching
mechanisms despite their high structural homology