Number Of Calcium Binding Sites Research Articles

Polymerization of sarcoplasmic-reticulum calcium-binding proteins might explain observed reticulum kinetics-on-demand behavior

Reported experimental results, in which transient elevations of sarcoplasmic calcium levels are induced by caffeine in smooth muscle cells, apparently contradict the principle of mass conservation. The commonly accepted model assumes that the total number of Ca2+ binding sites is fixed. A former work dealing with this problem proved that assuming the presence within the reticulum of calcium sequestering proteins whose total number of calcium binding sites increases as the existent sites get occupied, is enough to explain the above referred counter-intuitive experimental results. However, no chemical explanation was given to account for this binding-site count increase. In the present work, we propose a chemical-kinetics scheme for the binding of calcium to calsequestrin (a protein found within the reticulum) and the polymerization of this protein. On the one hand, this scheme is in agreement with reported results on calsequestrin binding kinetics, but it is also fully capable of explaining the observed intriguing performance of the sarcoplasmic reticulum. We further explore the behavior of the resulting nonlinear dynamic system and discuss possible physiological implications of the proposed scheme.

NTnC-like genetically encoded calcium indicator with a positive and enhanced response and fast kinetics

The NTnC genetically encoded calcium indicator has an advantageous design because of its smaller size, GFP-like N- and C-terminal ends and two-fold reduced number of calcium binding sites compared with widely used indicators from the GCaMP family. However, NTnC has an inverted and modest calcium response and a low temporal resolution. By replacing the mNeonGreen fluorescent part in NTnC with EYFP, we engineered an NTnC-like indicator, referred to as YTnC, that had a positive and substantially improved calcium response and faster kinetics. YTnC had a 3-fold higher calcium response and 13.6-fold lower brightness than NTnC in vitro. According to stopped-flow experiments performed in vitro, YTnC had 4-fold faster calcium-dissociation kinetics than NTnC. In HeLa cells, YTnC exhibited a 3.3-fold lower brightness and 4.9-fold increased response to calcium transients than NTnC. The spontaneous activity of neuronal cultures induced a 3.6-fold larger ΔF/F response of YTnC than previously shown for NTnC. On patched neurons, YTnC had a 2.6-fold lower ΔF/F than GCaMP6s. YTnC successfully visualized calcium transients in neurons in the cortex of anesthetized mice and the hippocampus of awake mice using single- and two-photon microscopy. Moreover, YTnC outperformed GCaMP6s in the mitochondria and endoplasmic reticulum of cultured HeLa and neuronal cells.

The number and location of EF hand motifs dictates the calcium dependence of polycystin‐2 function

Polycystin 2 (PC2) is a calcium-dependent calcium channel, and mutations to human PC2 (hPC2) are associated with polycystic kidney disease. The C-terminal tail of hPC2 contains 2 EF hand motifs, but only the second binds calcium. Here, we investigate whether these EF hand motifs serve as a calcium sensor responsible for the calcium dependence of PC2 function. Using NMR and bioinformatics, we show that the overall fold is highly conserved, but in evolutionarily earlier species, both EF hands bind calcium. To test whether the EF hand motif is truly a calcium sensor controlling PC2 channel function, we altered the number of calcium binding sites in hPC2. NMR studies confirmed that modified hPC2 binds an additional calcium ion. Single-channel recordings demonstrated a leftward shift in the calcium dependence, and imaging studies in cells showed that calcium transients were enhanced compared with wild-type hPC2. However, biophysics and functional studies showed that the first EF hand can only bind calcium and be functionally active if the second (native) calcium-binding EF hand is intact. These results suggest that the number and location of calcium-binding sites in the EF hand senses the concentration of calcium required for PC2 channel activity and cellular function.

Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes

The quality of genetically encoded calcium indicators (GECIs) has improved dramatically in recent years, but high-performing ratiometric indicators are still rare. Here we describe a series of fluorescence resonance energy transfer (FRET)-based calcium biosensors with a reduced number of calcium binding sites per sensor. These 'Twitch' sensors are based on the C-terminal domain of Opsanus troponin C. Their FRET responses were optimized by a large-scale functional screen in bacterial colonies, refined by a secondary screen in rat hippocampal neuron cultures. We tested the in vivo performance of the most sensitive variants in the brain and lymph nodes of mice. The sensitivity of the Twitch sensors matched that of synthetic calcium dyes and allowed visualization of tonic action potential firing in neurons and high resolution functional tracking of T lymphocytes. Given their ratiometric readout, their brightness, large dynamic range and linear response properties, Twitch sensors represent versatile tools for neuroscience and immunology.

Insights Into the Interaction of a Model Membrane Binding Protein, Annexin A5, with Calcium ion and Phospholipids Garnered via Isothermal Titration Calorimetry

Coupled equilibrium is a theme that runs throughout biochemical systems. It enables the ability to modulate a protein's response as a function of changing ligand levels. Isothermal titration calorimetry is a powerful tool to capture the thermodynamic signature of interactions as it relies upon the inherent heat of an interaction and not upon non-physiological probes. It is also a technique rift with potential complications due to the need of relatively high concentrations of macromolecule and ligand, but, more critically, only limited and somewhat simple models are readily available for analysis. Both of these issues are amplified in systems that contain acidic membrane and calcium ion, as precipitation is present and well developed binding models for membrane are not. The benefits, however, outweigh the potential for failure, and the resultant global analysis of the thermodynamic cycle of the interaction of a membrane-binding protein with phospholipid and calcium ion is presented. A single model was found to globally fit the interaction of annexin a5 with calcium ion, with calcium ion in the presence of phospholipid, with phospholipid in the presence of calcium ion, and annexin a5 with phospholipid. We find that the protein in the presence of phospholipid conserves its number of calcium-binding sites with near solution state affinity. In addition, binding phospholipid induces the formation of a number of new high affinity calcium sites; overall, this is a type of allosteric transition model. These low and high affinity calcium sites have very different calorimetric profiles. The model suggests that the small population of protein that binds membrane serves to thermodynamically position the protein such that it is able to bind calcium ion with much higher affinity than in solution.

Determination of calcium-binding constants of caseins, phosphoserine, citrate and pyrophosphate: A modelling approach using free calcium measurement

Dairy products contain large amount of calcium which is bound to caseins and different chelating agents like citrate and polyphosphates. The present study aimed to determine the calcium-binding capacities of phosphoserine (SerP), caseinophosphopeptide (CPP), β-casein, caseinate, citrate and pyrophosphate in the same conditions of temperature, pH and ionic strength. The free calcium (Ca2+) was measured using a calcium ion-selective electrode and plotted as a function of total calcium concentration. The association constants and the number of calcium-binding sites were determined by fitting the experimental data to a theoretical model. The phosphate groups of caseins were the main binding sites with evidence for participation of carboxylate groups. The intrinsic association constants determined by the best fit of the data were in the order: pyrophosphate (557×103M−1)>citrate (20×103M−1)>β-casein (5×103M−1)>caseinate, CPP and SerP (∼103M−1). These findings may be of interest for the development of calcium-enriched products to overcome calcium deficiency in specific populations.

Physicochemical Characterization of Casein Phosphopeptide-Amorphous Calcium Phosphate Nanocomplexes

Milk caseins stabilize calcium and phosphate ions and make them available to the neonate. Tryptic digestion of the caseins yields phosphopeptides from their polar N-terminal regions that contain clusters of phosphorylated seryl residues. These phosphoseryl clusters have been hypothesized to be responsible for the interaction between the caseins and calcium phosphate that lead to the formation of casein micelles. The casein phosphopeptides stabilize calcium and phosphate ions through the formation of complexes. The calcium phosphate in these complexes is biologically available for intestinal absorption and remineralization of subsurface lesions in tooth enamel. We have studied the structure of the complexes formed by the casein phosphopeptides with calcium phosphate using a range of physicochemical techniques including x-ray powder diffraction, scanning electron microscopy, transmission electron microscopy, and equilibrium binding analyses. The amorphous nature of the calcium phosphate phase was confirmed by two independent methods: x-ray powder diffraction and selected area diffraction. In solution, the ion activity product of a basic amorphous calcium phosphate phase was the only ion product that was a function of bound phosphate independent of pH, consistent with basic amorphous calcium phosphate being the phase stabilized by the casein phosphopeptides. Detailed investigations of calcium and calcium phosphate binding using a library of synthetic homologues and analogues of the casein phosphopeptides have revealed that although the fully phosphorylated seryl-cluster motif is pivotal for the interaction with calcium and phosphate, other factors are also important. In particular, calcium binding and calcium phosphate stabilization by the peptides was influenced by peptide net charge, length, and sequence.

PKC-induced Sensitization of Ca2+-dependent Exocytosis Is Mediated by Reducing the Ca2+ Cooperativity in Pituitary Gonadotropes

The highly cooperative nature of Ca2+-dependent exocytosis is very important for the precise regulation of transmitter release. It is not known whether the number of binding sites on the Ca2+ sensor can be modulated or not. We have previously reported that protein kinase C (PKC) activation sensitizes the Ca2+ sensor for exocytosis in pituitary gonadotropes. To further unravel the underlying mechanism of how the Ca2+ sensor is modulated by protein phosphorylation, we have performed kinetic modeling of the exocytotic burst and investigated how the kinetic parameters of Ca2+-triggered fusion are affected by PKC activation. We propose that PKC sensitizes exocytosis by reducing the number of calcium binding sites on the Ca2+ sensor (from three to two) without significantly altering the Ca2+-binding kinetics. The reduction in the number of Ca2+-binding steps lowers the threshold for release and up-regulates release of fusion-competent vesicles distant from Ca2+ channels.

Study of calcium binding to different preparations of yeast ( Saccharomyces cerevisiae) protein by using an ion selective electrode

The calcium binding capacity of a native brewer’s yeast protein (PL) was compared to protein from the same source, after treatment with 3% sodium trimetaphosphate (PLP). Calcium titration was done by the use of an ion-selective electrode. For measurements made at pH 6.5, the number of calcium binding sites per 10 5g protein was 29 for PL and 37 for PLP. The association constant K was 3.2×10 −3 and 2.7×10 −3 for PL and PLP, respectively. Dialysis of sample PLP against 0.01 M EDTA increased the number of binding sites to 49. The higher number of binding sites in PLP is explained by the presence of phosphate moieties associated with the protein molecules and also by possible conformational changes occurring in the protein molecules as a result of STMP treatment, exposing a larger number of amino acid side chains capable of combining with Ca 2+.©

Progress in electron microscopic diagnostics: semi‐quantitative determination of precipitable calcium in different cell types of the organ of Corti in the guinea‐pig

Potassium antimonate was used to precipitate calcium in the cochlea of the guinea-pig. The distribution of the calcium antimonate precipitates was analysed by electron microscopy. The precipitate density was determined in different cell types in the organ of Corti by counting the number of calcium binding sites in a 10-micron 2 area. The size of the precipitates varied considerably, and thus the relative amount of the precipitable calcium was estimated only semi-quantitatively. As the prominent carbon signal is superimposed over the nearby small Ca(2+)-edge signals, the combined signal of the antimony M4,5-edge and the oxygen K-edge of the calcium antimonate salt formed was chosen for the semi-quantitative estimation. Images of the inelastically scattered electrons of the precipitates at delta E = 570 eV were recorded by electron spectroscopic imaging. The area covered by the calcium precipitates within a given cell type was determined in different ultrathin sections of the same organ of Corti by an image processing system.

The effects of heating, alone or in the presence of calcium or lactose, on calcium binding to milk proteins

The binding of calcium by different milk proteins and the effects of heat were investigated. The influences of calcium and lactose were also investigated. It was shown that the caseins of cow's milk have a high calcium binding capacity, when treated with calcium at 25°C for 30 min, pH 7·00, due to the high number of calcium binding sites. The order of binding capacities is α s1 -casein > β-casein > κ-casein. For whey proteins the order is β-lactoglobulin (β-lg) > α-lactalbumin (α-la). Lactose, in general, when present at 25°C, did not appreciably affect the calcium binding of the major milk proteins. Heating of whole casein at 80 or 95°C for 30 min caused conformational changes of the protein resulting in an increase in its calcium binding ability. The presence of lactose during heating with or without calcium reduced its calcium binding. The calcium binding of α s1 -casein was not affected by heating, throughout the temperature range studied, either in the presence or in the absence of calcium, an indication that α s1 -casein may undergo physical changes at high temperatures, but the accessibility and/or the number of calcium binding centres are not increased. β-Casein, when heated with calcium, was subject to conformational changes which were reflected in changes in the degree of calcium-binding. Heating of β-lg in the presence of calcium did not much affect the amount of calcium bound to it. However, in the absence of calcium an increase of 21·5% in calcium binding was observed after heating at 80°C and a decrease of 40·0% after heating at 95°C, compared to unheated samples.

Effects of calcium on the stability and activity of guinea pig liver transglutaminase

The binding of calcium to transglutaminase was studied by a kinetic method and by spectrophotometric titration. By the first method, we have shown that the binding of a single calcium per molecule, with a binding constant of 7500 ± 1300 M −1 (at 55°C), was responsible for the enhancement of the thermostability. The kinetic constants of the deactivation for the unliganded native form and the liganded native one are 1.47 ± 0.04 min −1 and 0.32 ± 0.05 min −1 respectively. The enhancement of thermostability is due to the stabilization of the native form, since the deactivation constants of the liganded and unliganded intermediated forms are equal. The total number of calcium binding sites, determined by titration is 4, and therefore, only 1 of them should be implicated in the thermostability. The 4 apparent association constants have been determined by a non-linear fitting of the data to the Adair equation. We have also shown a positive co-operative behaviour of the transglutaminase when the transferase is monitored versus calcium concentration.

Primary structure of a zinc protease from Bacillus mesentericus strain 76

The amino acid sequence of the neutral zinc protease from Bacillus mesentericus strain 76 (MCP 76) has been determined by using peptides derived from digests with trypsin, chymotrypsin, and cyanogen bromide and from cleavage with o-iodosobenzoic acid. The peptides were purified by means of gel filtration and reversed-phase high-performance liquid chromatography and analyzed by automatic sequencing. The protein contains 300 amino acid residues. It proved to be identical with the neutral protease deduced from the DNA precursor sequence of Bacillus subtilis. The residues for zinc and substrate binding are conserved, whereas the number of calcium binding sites is reduced compared to thermolysin. A classification of the neutral zinc protease is discussed.

Calcium-induced associations of the caseins: A thermodynamic linkage approach to precipitation and resolubilization

Calcium-induced changes in protein solubility play a role in a variety of important biological processes including the deposition of bone and dentin and the secretion of milk. The phenomena of salt-induced (calcium) precipitation of proteins (salting-out), and the resolubilization of these proteins at higher salt concentrations (salting-in) have been studied and quantitated using an approach based on the concepts of Wyman's thermodynamic linkage. Salting-out has been described by a salt-binding constant, k 1, the number of moles of salt bound per mole of protein, n, and S 1, the fraction soluble at saturation of n; salting-in has been described by corresponding constants k 2, m, and S 2. Analysis of salt-induced solubility profiles was performed using nonlinear regression analysis. Results of calcium-induced solubility profiles of two genetic variants of α s1 -casein ( α s1 -A), ( α s1 -B), and β-casein C (β-C) at 37 °C, where hydrophobic interactions are maximized, showed no salting-in behavior and for salting-out, yielded k 1 values of 157, 186, and 156 liters · mol −1 and n values of 8, 8, and 4, respectively. The values of k 1 can be correlated with the apparent association constant for calcium binding to casein, while the values of n can be correlated with the number of calcium binding sites of the respective caseins. At 1 °C, where hydrophobic interactions are minimized, nominally only hydrophilic and electrostatic interactions can be linked to the salt-induced solubility profiles; here β-C is totally soluble at all calcium concentrations and α s1 -B and α s1 -A were now found to have salting-in parameters, K 2 and m, of 2.5 liters · mol −1 and 4, and 11 liters · mol −1 and 8, respectively. α s1 -A is more readily salted-in and studies on the variation of S 1 with added KCl for this protein at 1 °C indicated that salting-in is also mainly electrostatic in nature and may result from competition between K + and Ca 2+ for binding sites rather than from solute-solvent interactions as previously proposed. Comparison of k 1 and k 2 values between the two genetic variants, coupled with the known sequence differences (the A variant is a linear deletion of 13 amino acids) suggest the existence of a hydrophobically stabilized ion pair in α s1 -B which is deleted in α s1 -A; it is speculated that such bonds may play a role in other calcium-induced changes in protein Solubility.

Calcium binding by spinach stromal proteins

Calcium binding to spinach (Spinacia oleracea L.) stromal proteins was examined by dual-wavelength spectrophotometry using the metallochromic indicator tetramethylmurexide. The data are consistent with the existence of at least two, probably independent, classes of binding sites. The total number of binding sites varied between 90-155 nmol·mg(-1) protein with "average" binding constants of 1.1-2.7·mM(-1). Both Mg(2+) and La(3+) inhibited calcium binding competitively, with "average" inhibitor constants of 0.26·mM(-1) and 39.4·mM(-1), respectively; an increase in the potassium concentration up to 50 mM had no effect. In a typical experiment a decrease in pH (7.8 to 7.1) resulted in a decrease in the total number of calcium binding sites from 90 to 59 nmol·mg(-1) protein, but in an increase of the "average" affinity from 2.7 to 4.5·mM(-1). Calculations, using these data and those of Gross and Hess (1974, Biochim. Biophys. Acta 339, 334-346) for binding site I of washed thylakoid membranes, showed that the free-Ca(2+) concentration in the stroma under dark conditions, pH 7.1, is higher than under light conditions, pH 7.8. The physiological relevance of the observed calcium binding by stromal proteins is discussed.

Calcium binding to untreated and dephosphorylated porcine neurofilaments

The calcium-binding properties of untreated and in vitro dephosphorylated neurofilaments were determined by partition centrifugation. Scatchard plot analysis of the binding data indicated that each type of neurofilament contained both high and low affinity calcium-binding sites. The number of calcium-binding sites per unit consisting of three 140,000, three 107,000 and eight 62,000 molecular weight subunits was 4 sites with Kd=4.1 μM and 126 sites with Kd=293 μM for untreated neurofilaments. The in vitro dephosphorylated neurofilaments contained 8 sites with Kd=15 μM and 54 sites with Kd-332 μM, per unit.

Characterization of skeletal muscle calsequestrin by 1H NMR spectroscopy.

Calsequestrin (Mr = 44,000) is a calcium-binding (KD congruent to 1 mM, congruent to 50 sites/molecule) protein found in the lumen of the sarcoplasmic reticulum of skeletal muscle. The 1H NMR spectrum of calsequestrin in the calcium-free form is presented and is characteristic of a protein largely in the random coil configuration. A number of peaks in the aromatic region have been assigned based on their chemical shifts and sensitivity to pH. The interaction of this protein with Ca2+ and K+ was studied by 1H NMR. Potassium ion binding to calsequestrin caused broadening and concomitant loss of intensity in both the aromatic and aliphatic regions of the spectrum. Calcium ion binding caused similar effects but at much lower metal ion concentrations. It was found that the binding of Ca2+ to calsequestrin was cooperative (Hill coefficient n = 2.9 +/- 0.2) with a dissociation constant of 0.25 +/- 0.06 mM in the absence of K+. In contrast, K+ showed binding to a single class of independent sites (KD = 0.20 +/- 0.04 M). Calcium binding was also studied by circular dichroism at protein concentrations similar to the NMR experiments. The binding profile and cooperativity (n = 2.0 +/- 0.1, KD = 0.19 +/- 0.04 mM) were in agreement with the 1H NMR results. Circular dichroism studies performed at low protein concentrations to reduce the possible effect of calcium binding on the concentration of free calcium gave similar values of n = 2.42 +/- 0.14 and KD = 0.21 +/- 0.005 mM. This cooperativity was also observed in the presence of 100 mM KCl although the affinity for calcium has been significantly reduced (n = 1.65 +/- 0.09, KD = 0.87 +/- 0.036 mM). In view of the large number of calcium binding sites in calsequestrin, these small Hill coefficients show that calcium binding to calsequestrin is only mildly cooperative.

A conformational transition of the sarcoplasmic reticulum calcium transport ATPase induced by vanadate.

Vanadate binding to sarcoplasmic reticulum vesicles results in the loss of the externally located high affinity calcium binding sites of the calcium transport ATPase. Conversely the occupation by calcium of the internally located low affinity sites in the vanadate enzyme complex leads to the release of vanadate. Since the total number of calcium binding sites is not diminished by vanadate binding but slightly increases we conclude that vanadate binding induces a transition of the enzymes external high to internal low affinity calcium binding sites. The transposition of external to internal calcium binding sites is accompanied by a definite change in the structure of the sarcoplasmic reticulum membranes. On vanadate binding the asymmetrically arranged electron dense protein particles become symmetrically distributed.

Calcium binding of troponin C. I. A potentiometric titration study.

Structural changes of troponin C on calcium binding were studied by hydrogen ion titration, circular dichroism, and fluorescence measurements. The potentiometric titration curves in the carboxyl region are shifted towards lower pH with calcium binding. The intrinsic pK of the carboxyl groups at the calcium binding sites decreases by 0.8 pK unit on calcium binding; on the other hand, magnesium ions have little effect on the intrinsic pK of the carboxyl groups. The intrinsic pK of the imidazole group is not affected by calcium binding. The value of w, an electrostatic interaction factor, is identical for calcium-free and calcium-bound troponin C and is about half of the value calculated assuming a compact sphere. The results of difference titration on the calcium binding indicate that the pH of troponin C solution increases on addition of CaCl2 up to 2 mol of Ca2+ per mol of troponin C and then decreases on further addition of CaCl2. The pH increase is depressed in the presence of MgCl2, in the low pH region, or at high ionic strength. The pH increase is also observed on addition of MgCl2. The ellipticity at 222 nm was measured under the same conditions as the difference titration measurements, and the relation between the pH change and the conformational change of troponin C on calcium binding is discussed based on the results obtained. The number of calcium binding sites and the binding constants estimated by analysis of these difference titration curves were in agreement with the results of Potter and Gergely (22). No magnesium binding site was observed. The tyrosine fluorescence measurements indicated that the binding site near tyrosine-109 is one of the high affinity sites.

On the relation between filament overlap and the number of calcium-binding sites on glycerinated muscle fibers

The formation of rigor complexes between the thick and thin filaments of glycerinated rabbit psoas muscle fibers causes the fibers to bind more calcium at any given level of free calcium. I studied the maximum amount of calcium bound as a function of filament overlap under rigor conditions. Fibers stretched to zero filament overlap (sarcomere length greater than 3.8 micron) bound exactly 75% as much calcium as fibers with maximum overlap. Between these extremes a linear relationship was found between maximum bound calcium and the length of the overlap zone. The results support the hypothesis that in the intact filament lattice one of the four calcium-binding sites of troponin depends for its existence on attachment between myosin and actin. In addition, the linear relation between maximum bound calcium and filament overlap is consistent with the assumption that the cooperative effect of rigor complex formation on calcium binding is limited to the binding site in the immediate vicinity of the rigor complex.