Label-free quantification of calcium-sensor targeting to photoreceptor guanylate cyclase and rhodopsin kinase by backscattering interferometry.

Quantification of protein binding to membrane proteins is challenging and a limited set of methods is available to study such systems. Here we employed backscattering interferometry (BSI), a free-solution label-free method with high sensitivity, to quantify the interaction of neuronal Ca2+-Sensor proteins with their targets operating in phototransduction. We tested direct binding of guanylate cyclase-activating proteins (GCAP1 and GCAP2) to their membrane target guanylate cyclase 1. The regulatory mechanism of GCAPs including their binding interface in the target is unresolved. Here we used a label-free, free-solution assay method based on BSI to determine binding constants of GCAP1 and GCAP2 to the full-length membrane-bound guanylate cyclase type 1. GCAP1 and GCAP2 bound to different regions on the target guanylate cyclase with submicromolar affinity (apparent KD-values of 663 ± 121 nM and 231 ± 63 nM for Ca2+-free GCAP1 and GCAP2, respectively). A guanylate cyclase construct containing the juxta-membrane and kinase homology domain harbored an exclusive binding site for GCAP1 with similar affinities as the full-length protein, whereas GCAP2 did not bind to this region. We provide a model in which GCAP1 and GCAP2 do not share a single binding site to the target, thus cannot exchange upon fluctuating Ca2+ levels.

Bifunctional Diaminoterephthalate Fluorescent Dye as Probe for Cross-Linking Proteins.

Diaminoterephthalates are fluorescent dyes and define scaffolds, which can be orthogonally functionalized at their two carboxylate residues with functional residues bearing task specific reactive groups. The synthesis of monofunctionalized dyes with thiol groups for surface binding, an azide for click chemistry, and a biotinoylated congener for streptavidin binding is reported. Two bifunctionalized dyes were prepared: One with an azide for click chemistry and a biotin for streptavidin binding, the other with a maleimide for reaction with thiol and a cyclooctyne moiety for ligation with copper-free click chemistry. In general, the compounds are red to orange, fluorescent materials with an absorption at about 450 nm and an emission at 560 nm with quantum yields between 2-41 %. Of particular interest is the maleimide-functionalized compound, which shows low fluorescence quantum yield (2 %) by itself. After addition of a thiol, the fluorescence is "turned on"; quantum yield 41 %.

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.

Differential Nanosecond Protein Dynamics in Homologous Calcium Sensors.

Shaping the temporal response of photoreceptors is facilitated by a well-balanced second messenger cascade, in which two neuronal Ca(2+)-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(2+)-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(2+). 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.

Retina specific GCAPs in zebrafish acquire functional selectivity in Ca2+-sensing by myristoylation and Mg2+-binding.

Zebrafish photoreceptor cells express six guanylate cyclase-activating proteins (zGCAPs) that share a high degree of amino acid sequence homology, but differ in Ca(2+)-binding properties, Ca(2+)-sensitive target regulation and spatial-temporal expression profiles. We here study a general problem in cellular Ca(2+)-sensing, namely how similar Ca(2+)-binding proteins achieve functional selectivity to control finely adjusted cellular responses. We investigated two parameters of critical importance for the trigger and switch function of guanylate cyclase-activating proteins: the myristoylation status and the occupation of Ca(2+)-binding sites with Mg(2+). All zGCAPs can be myristoylated in living cells using click chemistry. Myristoylation does not facilitate membrane binding of zGCAPs, but it significantly modified the regulatory properties of zGCAP2 and zGCAP5. We further determined for all zGCAPs at least two binding sites exhibiting high affinities for Ca(2+) with KD values in the submicromolar range, whereas for other zGCAPs (except zGCAP3) the affinity of the third binding site was in the micromolar range. Mg(2+) either occupied the low affinity Ca(2+)-binding site or it shifted the affinities for Ca(2+)-binding. Hydrodynamic properties of zGCAPs are more influenced by Ca(2+) than by Mg(2+), although to a different extent for each zGCAP. Posttranslational modification and competing ion-binding can tailor the properties of similar Ca(2+)-sensors.

Structural effects of Mg²âº on the regulatory states of three neuronal calcium sensors operating in vertebrate phototransduction.

The effects of physiological concentration of magnesium on the switch states of the neuronal calcium sensor proteins recoverin, GCAP1 and GCAP2 were investigated. Isothermal titration calorimetry was applied for binding studies. Circular dichroism spectroscopy was used to characterize protein thermal stability, secondary and tertiary structure in conditions of high and low [Ca²âº], mimicking respectively the dark-adapted and light-exposed photoreceptor states during the phototransduction cascade. Further, molecular dynamics (MD) simulations were run to investigate the dynamical structural properties of GCAP1 in its activator, inhibitor and putative transitory states. Our results confirmed that Mg²âº is unable to trigger the typical Ca²âº-induced conformational change of recoverin (myristoyl switch) while it decreases its thermal stability. Interestingly, Mg²âº seems to affect the conformation of GCAP2 both at high and low [Ca²âº], however the variations are more substantial for myristoylated GCAP2 in the absence of Ca²âº. GCAP1 is responsive to Mg²âº only in its low [Ca²âº] state and Mg²âº-GCAP1 tertiary structure slightly differs from both apo and Ca²âº-bound states. Finally, MD simulations suggest that the GCAP1 state harboring one Mg²âº ion bound to EF2 acquires structural characteristics that are thought to be relevant for the activation of the guanylate cyclase. Moreover, all the putative Mg²âº-bound states of myristoylated GCAP1 are structurally less flexible than Ca²âº-bound states. GCAP1 acquires a more compact tertiary structure that is less accessible to the solvent, thereby inducing a different conformation to the myristoyl moiety, which might be crucial for the activation of the guanylate cyclase. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Conformational changes in calcium-sensor proteins under molecular crowding conditions.

Fundamental components of signaling pathways are switch modes in key proteins that control start, duration, and ending of diverse signal transduction events. A large group of switch proteins are Ca(2+) sensors, which undergo conformational changes in response to oscillating intracellular Ca(2+) concentrations. Here we use dynamic light scattering and a recently developed approach based on surface plasmon resonance to compare the protein dynamics of a diverse set of prototypical Ca(2+)-binding proteins including calmodulin, troponin C, recoverin, and guanylate cyclase-activating protein. Surface plasmon resonance biosensor technology allows monitoring conformational changes under molecular crowding conditions, yielding for each Ca(2+)-sensor protein a fingerprint profile that reflects different hydrodynamic properties under changing Ca(2+) conditions and is extremely sensitive to even fine alterations induced by point mutations. We see, for example, a correlation between surface plasmon resonance, dynamic light scattering, and size-exclusion chromatography data. Thus, changes in protein conformation correlate not only with the hydrodynamic size, but also with a rearrangement of the protein hydration shell and a change of the dielectric constant of water or of the protein-water interface. Our study provides insight into how rather small signaling proteins that have very similar three-dimensional folding patterns differ in their Ca(2+)-occupied functional state under crowding conditions.

Impact of cone dystrophy-related mutations in GCAP1 on a kinetic model of phototransduction.

Cone dystrophy-related mutations in guanylate cyclase-activating protein 1 (GCAP1) are known to cause severe disturbance of their Ca(2+)-sensing properties affecting also their regulatory modes. However, crucial biochemical properties of mutant GCAP1 forms have not been fully elucidated and regulatory parameters of GCAP1 mutants have not been considered within the context of a comprehensive description of the phototransduction cascade kinetics. We investigated therefore the structure-function relationships of four dystrophy-relevant point mutations in GCAP1 harboring the following amino acid substitutions: E89K, D100E, L151F, and G159V. All mutations decrease the catalytic efficiency in regulating the target guanylate cyclase and decrease the affinity of Ca(2+)-binding in at least one, but in most cases two EF-hand Ca(2+)-binding sites. Although the wild type and mutants of GCAP1 displayed large differences in Ca(2+)-binding and regulation, circular dichroism (CD) spectroscopy revealed that all proteins preserved an intact secondary and tertiary structure with a significant rearrangement of the aromatic residues upon binding of Ca(2+). To gain insight into the dynamic changes of cyclic GMP levels in a photoreceptor cell, we incorporated parameters describing the regulation of target guanylate cyclase by GCAP1 mutants into a comprehensive kinetic model of phototransduction. Modeling led us to conclude that the contribution of GCAP1 to the dynamic synthesis of cyclic GMP in rod cells would depend on the expression level of the wild-type form. Although the synthesis rate controlled by GCAP1 remains at a constant level, in the case of high expression levels of cone-dystrophy GCAP1 mutants it would not contribute at all to shaping the cGMP rate, which becomes dynamically regulated solely by the other present Ca(2+)-sensor GCAP2.

Dynamics of conformational Ca2+-switches in signaling networks detected by a planar plasmonic device.

Ca(2+)-sensor proteins regulate a variety of intracellular processes by adopting specific conformations in response to finely tuned changes in Ca(2+)-concentration. Here we present a surface plasmon resonance (SPR)-based approach, which allows for simultaneous detection of conformational dynamics of four Ca(2+)-sensor proteins (calmodulin, recoverin, GCAP1, and GCAP2) operating in the vertebrate phototransduction cascade, over variations in Ca(2+) concentration in the 0.1-0.6 µM range. By working at conditions that quantitatively mimic those found in the cell, we show that the method is able to detect subtle differences in the dynamics of each Ca(2+)-sensor, which appear to be influenced by the presence of free Mg(2+) at physiological concentration and by posttranslational modifications such as myristoylation. Comparison between the macroscopic Ca(2+)-binding constants, directly measured by competition with a chromophoric chelator, and the concerted binding-conformational switch detected by SPR at equilibrium reveals the relative contribution of the conformational change process to the SPR signal. This process appears to be influenced by the presence of other cations that perturb Ca(2+)-binding and the conformational transition by competing with Ca(2+), or by pure electrostatic screening. In conclusion, the approach described here allows a comparative analysis of protein conformational changes occurring under physiologically relevant molecular crowding conditions in ultrathin biosensor layers.