Experimental test of the probability density function of true value of Poisson distribution parameter by single observation of number of events

The empirical probability density function for the conditional distribution of the true value of Poisson distribution parameter on one measurement is constructed by computer experiment. The analysis of the obtained distributions confirms that these distributions are gamma-distributions.Comment: presented in the IX International Workshop on Advanced Computing and Analysis Techniques in Physics Research (ACAT'03), KEK, Tsukuba, Japan, December 1-5, 200

Synergetic effect of recoverin and calmodulin on regulation of rhodopsin kinase.

Phosphorylation of photoactivated rhodopsin by rhodopsin kinase (RK or GRK1), a first step of the phototransduction cascade turnoff, is under the control of Ca(2+)/recoverin. Here, we demonstrate that calmodulin, a ubiquitous Ca(2+)-sensor, can inhibit RK, though less effectively than recoverin does. We have utilized the surface plasmon resonance technology to map the calmodulin binding site in the RK molecule. Calmodulin does not interact with the recoverin-binding site within amino acid residues M1-S25 of the enzyme. Instead, the high affinity calmodulin binding site is localized within a stretch of amino acid residues V150-K175 in the N-terminal regulatory region of RK. Moreover, the inhibitory effect of calmodulin and recoverin on RK activity is synergetic, which is in agreement with the existence of separate binding sites for each Ca(2+)-sensing protein. The synergetic inhibition of RK by both Ca(2+)-sensors occurs over a broader range of Ca(2+)-concentration than by recoverin alone, indicating increased Ca(2+)-sensitivity of RK regulation in the presence of both Ca(2+)-sensors. Taken together, our data suggest that RK regulation by calmodulin in photoreceptor cells could complement the well-known inhibitory effect of recoverin on RK

Light-Induced Thiol Oxidation of Recoverin Affects Rhodopsin Desensitization

The excessive light illumination of mammalian retina is known to induce oxidative stress and photoreceptor cell death linked to progression of age-related macular degeneration. The photochemical damage of photoreceptors is suggested to occur via two apoptotic pathways that involve either excessive rhodopsin activation or constitutive phototransduction, depending on the light intensity. Both pathways are dramatically activated in the absence of rhodopsin desensitization by GRK1. Previously, we have shown that moderate illumination (halogen lamp, 1,500 lx, 1–5 h) of mammalian eyes provokes disulfide dimerization of recoverin, a calcium-dependent regulator of GRK1. Here, we demonstrate under in vivo conditions that both moderate long-term (metal halide lamp, 2,500 lx, 14 h, rat model) and intense short-term (halogen lamp, 30,000 lx for 3 h, rabbit model) illumination of the mammalian retina are accompanied by accumulation of disulfide dimer of recoverin. Furthermore, in the second case we reveal alternatively oxidized derivatives of the protein, apparently including its monomer with sulfinic group. Histological data indicate that thiol oxidation of recoverin precedes apoptosis of photoreceptors. Both disulfide dimer and oxidized monomer (or oxidation mimicking C39D mutant) of recoverin exhibit lowered α-helical content and thermal stability of their apo-forms, as well as increased Ca2+ affinity. Meanwhile, the oxidized monomer and C39D mutant of recoverin demonstrate impaired ability to bind photoreceptor membranes and regulate GRK1, whereas disulfide dimer exhibits notably improved membrane binding and GRK1 inhibition in absence of Ca2+. The latter effect is expected to slow down rhodopsin desensitization in the light, thereby favoring support of the light-induced oxidative stress, ultimately leading to photoreceptor apoptosis. Overall, the intensity and duration of illumination of the retina affect thiol oxidation of recoverin likely contributing to propagation of the oxidative stress and photoreceptor damage

Tuning of a neuronal calcium sensor

Recoverin is a Ca(2+)-regulated signal transduction modulator expressed in the vertebrate retina that has been implicated in visual adaptation. An intriguing feature of recoverin is a cluster of charged residues at its C terminus, the functional significance of which is largely unclear. To elucidate the impact of this segment on recoverin structure and function, we have investigated a mutant lacking the C-terminal 12 amino acids. Whereas in myristoylated recoverin the truncation causes an overall decrease in Ca(2+) sensitivity, results for the non-myristoylated mutant indicate that the truncation primarily affects the high affinity EF-hand 3. The three-dimensional structure of the mutant has been determined by x-ray crystallography. In addition to significant changes in average coordinates compared with wild-type recoverin, the structure provides strong indication of increased conformational flexibility, particularly in the C-terminal domain. Based on these observations, we propose a novel role of the C-terminal segment of recoverin as an internal modulator of Ca(2+) sensitivity

Zinc Modulation of Neuronal Calcium Sensor Proteins: Three Modes of Interaction with Different Structural Outcomes

International audienceNeuronal calcium sensors (NCSs) are the family of EF-hand proteins mediating Ca2+-dependent signaling pathways in healthy neurons and neurodegenerative diseases. It was hypothesized that the calcium sensor activity of NCSs can be complemented by sensing fluctuation of intracellular zinc, which could further diversify their function. Here, using a set of biophysical techniques, we analyzed the Zn2+-binding properties of five proteins belonging to three different subgroups of the NCS family, namely, VILIP1 and neurocalcin-δ/NCLD (subgroup B), recoverin (sub-group C), as well as GCAP1 and GCAP2 (subgroup D). We demonstrate that each of these proteins is capable of coordinating Zn2+ with a different affinity, stoichiometry, and structural outcome. In the absence of calcium, recoverin and VILIP1 bind two zinc ions with submicromolar affinity, and the binding induces pronounced conformational changes and regulates the dimeric state of these proteins without significant destabilization of their structure. In the presence of calcium, recoverin binds zinc with slightly decreased affinity and moderate conformational outcome, whereas VILIP1 becomes 2+ 2+ insensitive to Zn . NCALD binds Zn with micromolar affinity, but the binding induces dramatic destabilization and aggregation of the protein. In contrast, both GCAPs demonstrate low-affinity binding of zinc independent of calcium, remaining relatively stable even at submillimolar Zn2+ concentrations. Based on these data, and the results of structural bioinformatics analysis, NCSs can be divided into three categories: (1) physiological Ca2+/Zn2+ sensor proteins capable of binding exchangeable (signaling) zinc (recoverin and VILIP1), (2) pathological Ca2+/Zn2+ sensors responding only to aberrantly high free zinc concentrations by denaturation and aggregation (NCALD), and (3) Zn2+-resistant, Ca2+ sensor proteins (GCAP1, GCAP2). We suggest that NCS proteins may there- fore govern the interconnection between Ca2+ dependent and Zn2+-dependent signaling pathways in healthy neurons and zinc cytotoxicity-related neurodegenerative diseases, such as Alzheimer’s disease and glaucoma