Affinity Calcium Binding Research Articles

Load dependence of ventricular performance explained by model of calcium-myofilament interactions.

Although a simple concept of load-independent behavior of the intact heart evolved from early studies of isolated, intact blood-perfused hearts, more recent studies showed that, as in isolated muscle, the mode of contraction (isovolumic vs. ejection) impacts on end-systolic elastance. The purpose of the present study was to test whether a four-state model of myofilament interactions with length-dependent rate constants could explain the complex contractile behavior of the intact, ejecting heart. Studies were performed in isolated, blood-perfused canine hearts with intracellular calcium transients measured by macroinjected aequorin. Measured calcium transients were used as the driving function for the model, and length-dependent rate constants yielding the highest concordance between measured and model-predicted midwall stress at different isovolumic volumes were determined. These length-dependent rate constants successfully predicted contractile behavior on ejecting contractions. This, along with additional model analysis, suggests that length-dependent changes in calcium binding affinity may not be an important factor contributing to load-dependent contractile performance in the intact heart under physiological conditions.

Cyclic octapeptides containing thiazole. Effect of stereochemistry and degree of flexibility on calcium binding propertiesElectronic supplementary information (ESI) available: NOE and CD titration data for 5. See http://www.rsc.org/suppdata/p2/b1/b109168a/

Solution conformation and calcium binding properties have been investigated for the two cyclic octapeptides cyclo(-D-Thr-D-Val(Thz)-Ile-)2 (4) and cyclo(-Thr-Gly(Thz)-Ile-Ser-Gly(Thz)-Ile-) (5) and the results are compared to those for the cyclic octapeptides previously studied; ascidiacyclamide (1), patellamide D (2), cyclo(-Thr-D-Val(Thz)-Ile-)2 (3), and cyclo(-Thr-D-Val-αAbu-Ile-)2 (6). Both 4 and 5 contain two heterocyclic thiazole ring constraints but the latter has a larger degree of flexibility as a consequence of the glycine residues within the cyclic framework. The solution conformation of 4 and 5 was determined from 1H NMR spectra and found to be a “twisted figure of eight” similar to that for 2. Complexation studies using 1H NMR and CD spectroscopy yielded 1 ∶ 1 calcium–peptide binding constants (logK) for the two peptides (2.3 (4) and 5.7 (5)). For 5 the magnitude of the binding constant was verified by a competition titration using CD. The different calcium-binding affinities of 3 (logK = 4.0) and 4 is attributed to the stereochemistry of the threonine residue. The magnitude of the binding constant for 5 compared to 3 and 4 (all peptides containing two thiazole ring constrains) demonstrates that the increase in flexibility of the cyclic peptide has a dramatic effect on the Ca2+ binding ability. The affinity for Ca2+ thus decreases in the order (6 ∼ 5 > 3 > 2 ∼ 1 > 4). The number of carbonyl donors available on each peptide has only a limited effect on calcium binding. The most important factor is the flexibility, which allows for a conformation of the peptide capable of binding calcium efficiently.

PH-dependent regulation of lysosomal calcium in macrophages.

Calcium measurements in acidic vacuolar compartments of living cells are few, primarily because calibration of fluorescent probes for calcium requires knowledge of pH and the pH-dependence of the probe calcium-binding affinities. Here we report pH-corrected measurements of free calcium concentrations in lysosomes of mouse macrophages, using both ratiometric and time-resolved fluorescence microscopy of probes for pH and calcium. Average free calcium concentration in macrophage lysosomes was 4-6x10(-4) M, less than half of the extracellular calcium concentration, but much higher than cytosolic calcium levels. Incubating cells in varying extracellular calcium concentrations did not alter lysosomal pH, and had only a modest effect on lysosomal calcium concentrations, indicating that endocytosis of extracellular fluid provided a small but measurable contribution to lysosomal calcium concentrations. By contrast, increases in lysosomal pH, mediated by either bafilomycin A(1) or ammonium chloride, decreased lysosomal calcium concentrations by several orders of magnitude. Re-acidification of the lysosomes allowed rapid recovery of lysosomal calcium concentrations to higher concentrations. pH-dependent reductions of lysosomal calcium concentrations appeared to result from calcium movement out of lysosomes into cytoplasm, since increases in cytosolic calcium levels could be detected upon lysosome alkalinization. These studies indicate that lysosomal calcium concentration is high and is maintained in part by the proton gradient across lysosomal membranes. Moreover, lysosomes could provide an intracellular source for physiological increases in cytosolic calcium levels.

Design, synthesis, and characterization of a calcium-sensitive near infrared dye

Intracellular calcium concentration in biological cells varies from 0.1 to 10 μM depending upon cell signaling and disease states. A direct estimate of calcium concentration in cell tissues within this range is possible with a novel calcium-selective reagent 15C5-774. The molecule of 15C5-774 consists of a near-infrared (NIR) chromophore ( λ max=774 nm) and a metal complexing moiety of benzo-15-crown-5. The reagent shows a strong calcium binding affinity in a 1:1 ratio and metal selectivity in the order Ca 2+>Mg 2+>Sr 2+≈K +≈Na +>Zn 2+>Li +. The high sensitivity is achieved by conducting absorption measurements in the NIR region where background interference from the biological matrix is low.

Metal binding affinity and structural properties of an isolated EF-loop in a scaffold protein

To establish an approach to obtain the site-specific calcium binding affinity of EF-hand proteins, we have successfully designed a series of model proteins, each containing the EF-hand calcium-binding loop 3 of calmodulin, but with increasing numbers of Gly residues linking the loop to domain 1 of CD2. Structural analyses, using different spectroscopic methods, have shown that the host protein is able to retain its native structure after insertion of the 12-residue calcium-binding loop and retains a native thermal stability and thermal unfolding behavior. In addition, calcium binding to the engineered CD2 variants does not result in a significant change from native CD2 conformation. The CD2 variant with two Gly linkers has been shown to have the strongest metal binding affinity to Ca(II) and La(III). These experimental results are consistent with our molecular modeling studies, which suggest that this protein with the engineered EF-loop has a calmodulin-like calcium binding geometry and backbone conformation. The addition of two Gly linkers increases the flexibility of the inserted EF-loop 3 from calmodulin, which is essential for the proper binding of metal ions.

Mutating aspartate in the calcium-binding site of alpha-lactalbumin: effects on the protein stability and cation binding.

The residue Asp87, which is in the calcium-binding loop of bovine alpha-lactalbumin (alpha-LA) and provides a side-chain carboxylate oxygen for ligand Ca(II) co-ordination, was substituted by either alanine or asparagine. The physical properties and calcium-binding affinities were monitored by intrinsic fluorescence and circular dichroism spectroscopy. D87A alpha-LA displayed a total loss of rigid tertiary structure, a dramatic loss in secondary structure and negligible calcium affinity [Anderson et al. (1997) Biochemistry, 36, 11648-11654]. On the contrary, D87N alpha-LA displayed native-like secondary structure with a somewhat de-stabilized tertiary structure. When the well-documented N-terminal methionine was enzymatically removed from D87N alpha-LA [Veprintsev et al. (1999) PROTEINS: Struct. Funct. Genet., 37, 65-72], the structure appeared to more closely resemble native alpha-LA. Remarkably, the thermal transition mid-temperature of apo-desMetD87N alpha-LA was approximately 31 degrees C versus native apo- alpha-LA (approximately 25 degrees C), probably due to negative charge 'compensation' in the calcium co-ordination site. On the other hand, the transition mid-temperature of Ca(II)-bound desMetD87N alpha-LA was approximately 57 degrees C versus native alpha-LA (approximately 66 degrees C), which was related to a decreased Ca(II) affinity (K = approximately 2.1 x 10(5) versus approximately 1.7 x 10(7)/M at 40 degrees C, respectively). These results reaffirm that alanine substitution in site specific mutagenesis is not always a prudent choice. Substitutions must be conservative with only minimal changes in functional groups and side-chain volume.

Calcium-regulated DNA binding and oligomerization of the neuronal calcium-sensing protein, calsenilin/DREAM/KChIP3.

Calsenilin/DREAM/KChIP3, a member of the recoverin branch of the EF-hand superfamily, interacts with presenilins, serves as a calcium-regulated transcriptional repressor, and interacts with A-type potassium channels. Here we report physicochemical characterization of calcium binding, oligomerization, and DNA binding of human calsenilin/DREAM/KChIP3. Equilibrium Ca(2+) binding measurements indicate that the protein binds 3 Ca(2+) with a dissociation constant of 14 microM and a Hill coefficient of 0.7. Dynamic light scattering and size exclusion chromatography show that the Ca(2+)-bound protein exists as a dimer at protein concentrations lower than 150 microM and forms a tetramer at concentrations above 200 microM. The Ca(2+)-free protein is a tetramer in the concentration range 20-450 microM. Isothermal titration calorimetry and dynamic light scattering indicate that the Ca(2+)-free protein tetramer binds endothermically (DeltaH = +25 kcal/mol) to four molecules of DNA derived from the downstream regulatory element (DRE) of either the prodynorphin or c-fos genes. One DRE molecule binds tightly to the protein with a dissociation constant (K(d)) of 75 nM, and the other three bind more weakly (K(d) = 640 nM). No significant DNA binding was observed for the Ca(2+)-bound protein. The N-terminal protein fragment (residues 1-70) binds nonspecifically to DRE in a Ca(2+)-independent manner, whereas a C-terminal fragment containing the four EF-hands (residues 65-256) binds DRE (K(d) = 200 nM) in a Ca(2+)-regulated and sequence-specific fashion. The C-terminal fragment is a tetramer in the Ca(2+)-free state and dissociates into dimers at saturating Ca(2+) levels.

Contribution of potential EF hand motifs to the calcium-dependent gating of a mouse brain large conductance, calcium-sensitive K(+) channel.

1. The large conductance, calcium-sensitive K(+) channel (BK(Ca) channel) is a unique member of the K(+)-selective ion channel family in that activation is dependent upon both direct calcium binding and membrane depolarization. Calcium binding acts to dynamically shift voltage-dependent gating in a negative or left-ward direction, thereby adjusting channel opening to changes in cellular membrane potential. 2. We hypothesized that the intrinsic calcium-binding site within the BK(Ca) channel alpha subunit may contain an EF hand motif, the most common, naturally occurring calcium binding structure. Following identification of six potential sites, we introduced a single amino acid substitution (D/E to N/Q or A) at the equivalent of the -z position of a bona fide EF hand that would be predicted to lower calcium binding affinity at each of the six sites. 3. Using macroscopic current recordings of wild-type and mutant BK(Ca) channels in excised inside-out membrane patches from HEK 293 cells, we observed that a single point mutation in the C-terminus (Site 6, FLD(923)QD to N), adjacent to the 'calcium bowl' described by Salkoff and colleagues, shifted calcium-sensitive gating right-ward by 50--65 mV over the range of 2--12 microM free calcium, but had little effect on voltage-dependent gating in the absence of calcium. Combining this mutation at Site 6 with a similar mutation at Site 1 (PVD(81)EK to N) in the N-terminus produced a greater shift (70--90 mV) in calcium-sensitive gating over the same range of calcium. We calculated that these combined mutations decreased the apparent calcium binding affinity approximately 11-fold (129.5 microM vs. 11.3 microm) compared to the wild-type channel. 4. We further observed that a bacterially expressed protein encompassing Site 6 of the BK(Ca) channel C-terminus and bovine brain calmodulin were both able to directly bind (45)Ca(2+) following denaturation and polyacrylamide gel electrophoresis (e.g. SDS-PAGE). 5. Our results suggest that two regions within the mammalian BK(Ca) channel alpha subunit, with sequence similarities to an EF hand motif, functionally contribute to the calcium-sensitive gating of this channel.

Peptide analogs from E-cadherin with different calcium-binding affinities.

Cadherins are a family of calcium-dependent cell-surface proteins that are fundamental in controlling the development and maintenance of tissues. Motif B of E-cadherin seems to be a crucial calcium-binding site as single point mutations (D134A and D134K) completely inactivate its adhesion activity. We analyzed peptide models corresponding to motif B (amino acids 128-144) as well as selected mutations of this motif. Our NMR studies showed that this motif B sequence is actually an active calcium-binding region, even in the absence of the rest of the cadherin molecule. We found that the binding affinity of this motif is very sensitive to mutations. For example, our peptide P128-144 with the native calcium-binding sequence has an affinity of Kd 0.4 mM, whereas the mutants P128-144/ D134A and P128-144/D134K containing the replacement of Asp134 by Ala and Lys, have Kd values of only 1.5 and 11 mM, respectively. Removing Asp at position 134, which correlates with the loss of adhesion activity, decreases calcium-binding affinity 20-fold. Ala132, along with residues Asp134, Asp136 and Asn143, is involved in calcium binding in solution. We also demonstrated that the calcium-binding affinity can be increased 3-fold when an additional Asp is introduced at position 132. In 50% organic solvent, this binding affinity of peptide P128-144/A132D (17-mer) from E-cadherin is similar to that of peptide P72-100/C73-77-91A (29-mer) from alpha-lactalbumin.

The mitochondrial permeability transition pore.

This chapter reviews recent advances in the identification of the structural elements of the permeability transition pore. The discovery that cyclosporin A (CsA) inhibits the pore proved instrumental. Various approaches indicate that CsA blocks the pore by binding to cyclophilin (CyP)-D. In particular, covalent labelling of CyP-D in situ by a photoactive CsA derivative has shown that pore ligands have the same effects on the degree to which CsA both blocks the pore and binds to CyP-D. The recognition that CyP-D is a key component has enabled the other constituents to be resolved. Use of a CyP-D fusion protein as affinity matrix has revealed that CyP-D binds very strongly to 1:1 complexes of the voltage-dependent anion channel (from the outer membrane) and adenine nucleotide translocase (inner membrane). Our current model envisages that the pore arises as a complex between these three components at contact sites between the mitochondrial inner and outer membranes. This is in line with recent reconstitutions of pore activity from protein fractions containing these proteins. The strength of interaction between these proteins suggests that it may be a permanent feature rather than assembled only under pathological conditions. Calcium, the key activator of the pore, does not appear to affect pore assembly; rather, an allosteric action allowing pore flicker into an open state is indicated. CsA inhibits pore flicker and lowers the binding affinity for calcium. Whether adenine nucleotide translocase or the voltage-dependent anion channel (via inner membrane insertion) provides the inner membrane pore has not been settled, and data relevant to this issue are also documented.

Identification of calcium binding sites in the trypanosome flagellar calcium-acyl switch protein

The 24 kDa flagellar calcium binding protein (FCaBP) of the protozoan Trypanosoma cruzi is a calcium-acyl switch protein. FCaBP is modified by the addition of myristate and palmitate at its amino terminal segment and both modifications are required for calcium-modulated flagellar membrane association. FCaBP has four sequence motifs for potential calcium binding, and comparison to other calcium-acyl switch proteins, such as recoverin, suggested that only two of these sites are functional. Because it is not possible to predict with certainty the calcium binding affinity or selectivity based on motif analysis alone, we determined the quantitative calcium binding activity of FCaBP by direct ligand binding using the flow dialysis method. The results demonstrated the presence of two calcium binding sites in the full length FCaBP and in a mutant (FCaBPΔ12) lacking the amino terminal pair of sites. FCaBPΔ12 retains its ability to localize to the flagellum. A mutant FCaBP lacking the two carboxyl-terminal sites (FCaBPΔ34), did not bind calcium with high affinity and selectivity under the conditions used. The calcium binding properties of FCaBP are therefore distinct from other myristoyl switch proteins such as recoverin. The results add to a growing body of knowledge about the correlation of sequence motifs with calcium binding activity. Moreover, they demonstrate the need to determine the apparently novel mechanism by which FCaBP undergoes calcium modulated flagellar membrane association and its relation to calcium signal transduction.

Coexpression of neurocalcin with other calcium-binding proteins in the rat main olfactory bulb.

The distribution patterns of four calcium-binding proteins (CaBPs)-calbindin D-28k (CB), calretinin (CR), neurocalcin (NC), and parvalbumin (PV)-in the rat main olfactory bulb were compared, and the degrees ofcolocalization of NC with the other CaBPs were determined by using double immunocytochemical techniques. All investigated CaBPs were detected in groups of periglomerular cells and Van Gehuchten cells, whereas other cell types expressed some of the investigated proteins but not all four. Double-labeling techniques demonstrated the colocalization of NC with CB, CR, or PV in periglomerular cells, whereas each neurochemical group constituted entirely segregated populations in the remaining neuronal types. This is evident in granule cells that demonstrated large but segregated populations immunoreactive to either NC or CR. This study provides a further biochemical characterization of interneuronal types in the rat main olfactory bulb. On the basis of the distinct calcium-binding affinities, each neurochemically defined population may have different responses to calcium influx that would result in the existence of distinct functional subgroups within morphologically defined neuronal types. The expression of the investigated CaBPs in periglomerular cells with both single and colocalized patterns suggests that the local circuits in the glomerular layer are constituted by a complex network of elements with particular calcium requirements.

EGF-like domain calcium affinity modulated by N-terminal domain linkage in human fibrillin-1

Calcium binding epidermal growth factor-like domains (cbEGFs) are present in many extracellular proteins, including fibrillin-1, Notch-3, protein S, factor IX and the low density lipoprotein (LDL) receptor, which perform a diverse range of functions. Genetic mutations that cause amino acid changes within these proteins have been linked to the Marfan syndrome (MFS), CADASIL, protein S deficiency, haemophilia B and familial hypercholesterolaemia, respectively. A number of these mutations disrupt calcium binding to cbEGFs, emphasising the critical functional role of calcium in these proteins.We have determined the calcium binding affinity of two sites within a cbEGF pair (cbEGF12-13) from human fibrillin-1 using two-dimensional nuclear magnetic resonance (NMR) and fluorescence techniques. Fibrillin-1 is a mosaic protein containing 43 cbEGF domains, mainly arranged as tandem repeats. Our results show that the cbEGF13 site in the cbEGF12-13 pair possesses the highest calcium affinity of any cbEGF investigated from fibrillin-1. A comparative analysis of these and previously reported calcium binding data from fibrillin-1 demonstrate that the affinity of cbEGF13 is enhanced more than 70-fold by the linkage of an N-terminal cbEGF domain. In contrast, comparison of calcium binding by cbEGF32 in isolation relative to when linked to a transforming growth factor β-binding protein-like domain (TB6-cbEGF32) reveals that the same enhancement is not observed for this heterologous domain pair. Taken together, these results indicate that fibrillin-1 cbEGF Ca2+affinity can be significantly modulated by the type of domain which is linked to its N terminus. The cbEGF12-13 pair is located within the longest contiguous section of cbEGFs in fibrillin-1, and a number of mutations in this region are associated with the most severe neonatal form of MFS. The affinities of cbEGF domains 13 and 14 in this region are substantially higher than in the C-terminal region of fibrillin-1. This increased affinity may be important for fibrillin assembly into 10–12 nm connective tissue microfibrils and/or may contribute to the biomechanical properties of the microfibrillar network.

Fluoride-Induced Enhancement of Diffusion in Streptococcal Model Plaque Biofilms

It has been demonstrated that fluoride decreases the calcium-binding affinity of Streptococcus mutans and approximately doubles the calcium-binding capacity. To investigate the effect of this mechanism on calcium mobility in plaque, ⁴⁵Ca flux was measured from a condensed films of S. mutans into tracer-free solution. Bacteria were suspended in pH 7.0 or 5.0 buffer including 0, 5, 10, 15 or 20 mmol/l Ca²⁺ carrier, with or without 5 mmol/l F^– and with ⁴⁵Ca and ³H-inulin. The appearance of ⁴⁵Ca and ³H-inulin in carrier-containing but initially tracer-free buffer was measured and extracellular fraction (V_e) and bound calcium were calculated. As the ratio (R) of bound to free Ca²⁺ approached zero at high [Ca²⁺], the measured diffusion coefficient (_rD_e) approached the effective diffusion coefficient (D_e), such that: _rD_e = D_e/(1+R). Fluoride increased the rate of calcium diffusion by a reduction in the binding affinity. This work demonstrates that fluoride significantly increases mobility in plaque; this may increase the rate at which calcium is transported between plaque and an underlying lesion and so promote remineralization. This mechanism could also increase the penetration of bacteriocides and suggests a novel method for biofilm treatment.

Calcium induces a conformational change in the ligand binding domain of the low density lipoprotein receptor

We previously have shown that LDL-R354, a truncated low density lipoprotein (LDL) receptor, is a calcium binding protein. LDL-R354 is composed of the ligand binding domain and repeat A of the EGF precursor homology domain of the full-length human LDL receptor. We also found that Ca2+ was required for the interaction between LDL-R354 and its ligand, LDL (Dirlam et al. 1996. Protein Expr. Purif.8: 489–500). In the current study, calcium-induced changes in the structure and function of LDL-R354 were examined. When calcium bound to LDL-R354, its apparent size increased, as determined by native and SDS-gel electrophoresis. The calcium-saturated form of LDL-R354 was more resistant to trypsin proteolysis than the calcium-depleted form. In the presence of calcium, the disulfide bonds in the truncated receptor were stabilized, rendering them more resistant to reduction by dithiothreitol. Calcium binding affinities were measured by monitoring increased tryptophan fluorescence intensities. LDL-R354 bound Ca2+ with high affinity (EC50 = 60 nm at pH 7.4) and specificity, as 400 μm Mg2+ did not compete for calcium binding. The affinity of LDL-R354 for calcium decreased when the pH was lowered. These results suggest that calcium induces a conformational change in the ligand binding domain of the LDL receptor and that a receptor conformer capable of binding ligand should be stabilized at physiological extracellular Ca2+ concentration and pH. Drops in pH may regulate LDL receptor function by altering the amount of calcium bound to the receptor.—Dirlam-Schatz, K. A., and A. D. Attie. Calcium induces a confirmation change in the ligand binding domain of the low density lipoprotein receptor.

Functional identification of calcium binding residues in bovine alpha-lactalbumin.

The functional role of previously identified calcium binding residues in alpha-lactalbumin (alpha-LA) was investigated by site-directed mutagenesis. Mutation of D82 to alanine did not effect the binding affinity for calcium, the protein structure, or its function in the lactose synthase assay, suggesting that this aspartate side chain is not essential for calcium binding or structural stabilization. In contrast, mutation of either D87 or D88 to alanine completely eliminated the strong calcium binding and altered alpha-LA as shown by several spectroscopically derived properties such as near- and far-UV CD and intrinsic fluorescence studies. These latter two mutants displayed significantly reduced abilities to stimulate lactose synthase activity (<3.5% of the maximal rate). Additionally, residues K79 and D84, which chelate calcium by backbone carbonyls, were mutated to alanine. K79A lost approximately 50% of its tertiary structure and stability (as determined by CD) but retained full calcium binding activity, indicating that at least the lysine side chain does not influence the carbonyl-mediated calcium coordination. In contrast, D84A lost approximately 25% of its tertiary structure and stability which was accompanied by a modest reduction in calcium affinity. Both mutants were able to stimulate normal lactose synthase activity. The triple mutant, D82A/D87A/D88A alpha-LA, lost its ability to bind calcium, similar to D87A and D88A. These studies clearly demonstrate the importance and variation of side chain interactions, which might be the seminal event in the establishment of the correct calcium binding loop conformation, possibly to stabilization and final folding of the overall protein structure.

Structure/calcium affinity relationships of site III of calmodulin: testing the acid pair hypothesis using calmodulin mutants.

Calmodulin mutants in which the calcium binding affinity of site IV was greatly reduced by a D133E mutation were prepared using site-specific, cassette-mediated mutagenesis as a multisite calcium binding protein model to examine structure/calcium affinity relationships in site III of calmodulin. Tryptophan was introduced in position 92 of the calmodulin mutants as a fluorescent label to monitor the calcium-induced structural changes in the C-terminal domain of calmodulin. The five calmodulin mutants, 3xCaM, 3zCaM, 4xCaM, 4zCaM, and 4xzCaM, were designed so that there were three or four acidic amino acid residues in chelating positions of site III with acid pairs on either the X and/or Z coordinating axes. The calcium dissociation constant of site III, KIII, of the five calmodulin mutants changes in a descending order from 3xCaM (237 microM), 3zCaM (140 microM), 4xCaM (5.8 microM), 4zCaM (3 microM), to 4xzCaM (2 microM), and these KIII values are significantly lower than that of F92W/D133E calmodulin (335 microM) in which three acidic residues with no acid pairs were present in site III [Wu, X., & Reid, R. E. (1997) Biochemistry 36, 3608-3616]. These results indicate that the calcium affinity of site III increases when the number of the acidic chelating residues increases from three to four, when the number of acid pairs increases from zero to one and further to two, and when the location of the acid pair is changed from the X axis to the Z axis. This study provides the first evidence that the acid pair hypothesis which correlates the nature of the chelating residues with the calcium affinity of the hlh motif is applicable to a multisite calcium binding protein model. The Hill coefficients indicate that reversal of the sequence of filling of the calcium binding sites in the C-terminal domain from IV --> III to III --> IV also changes the site cooperativity from positive to negative. The cooperativity returns to positive when the proteins are titrated in the presence of a calmodulin-binding peptide. Data from the present study also demonstrate that calmodulin mutants with a decreased calcium affinity have a reduced efficiency in phosphodiesterase regulation at low calcium concentrations (50 microM). However, high calcium concentrations (15 mM) restore the phosphodiesterase regulatory activity of the calmodulin mutants to a level obtained with F92W calmodulin, indicating that the mutations alter calcium regulation of calmodulin-mediated phosphodiesterase activity without affecting the interaction between calmodulin and the enzyme.

Eggshells are shaped by a precise spatio-temporal arrangement of sequentially deposited macromolecules.

The avian eggshell is a composite bioceramic which is formed by a controlled interaction of an organic and an inorganic phase. The organic phase contains, among other constituents, type X collagen and proteoglycans, mainly keratan and dermatan sulfate. Understanding the principles governing the synthesis and temporo-spatial distribution of such macromolecules, and their influence on the organization of the crystalline phase, is an essential aspect of establishing the biological basis of the quality of eggshell, both as an embryonic chamber and as a natural food package. In the present study, we have examined the process of eggshell formation by immunohistochemistry, scanning electron microscopy and energy dispersive X-ray microanalysis. Precise sites and timing of secretion were established for the deposition of particular macromolecules. Type X collagen is detected at the very first moment of shell membrane formation. The appearance of keratan sulfate coincides with the appearance of mammillae, while dermatan sulfate is deposited later, coincident with shell matrix deposition. We propose that keratan sulfate, due to its precise localization, temporal appearance and calcium-binding affinity, relates to the maintenance of calcium reserve bodies, the primary source of calcium for the embryo. On the other hand, dermatan sulfate may control crystal growth, resulting in a preferential orientation of calcite crystals within the palisade layer.

Conservative D133E Mutation of Calmodulin Site IV Drastically Alters Calcium Binding and Phosphodiesterase Regulation

Two calmodulin mutants, F92W and F92W/D133E, were prepared using site-specific cassette-mediated mutagenesis to examine the structure/calcium affinity relationships of cation chelating residues in calcium binding sites III and IV. The mutant, F92W, was prepared to produce a strong fluorescent label to follow the calcium-induced structural changes in the C-terminal domain of the protein. A second mutant, F92W/D133E, was prepared to destroy the calcium binding to site IV and thereby eliminate cooperativity between sites III and IV. The macroscopic calcium dissociation constants of the two sites in the C-terminal domain were derived from the calcium titration data that had been fitted to a two-site Hill equation. The calcium dissociation constants of site III and site IV in the F92W/D133E mutant were 335 microM and 2.76 mM, respectively. These values were significantly greater than the values of 14 and 1 microM for site III and site IV in F92W calmodulin, respectively. These results suggested that a very conservative D133E mutation in the +Z position of the site IV Ca2+-binding loop drastically decreased the calcium binding affinity of the site (2760-fold) and also significantly reduced that of site III in the same domain (24-fold). The D/E calmodulin mutant also had a 3-fold lower phosphodiesterase activation activity with a 25-fold lower affinity for this enzyme than that of F92W calmodulin in the presence of low calcium concentration (50 microM). However, the maximum phosphodiesterase activation activity of the F92W/D133E mutant and the affinity of this mutant for the enzyme were similar to those of F92W calmodulin in the presence of high calcium concentration (15 mM), suggesting that the D133E mutation altered calcium regulation of calmodulin mediated phosphodiesterase activity without affecting calmodulin interaction with the enzyme.

Modified helix-loop-helix motifs of calmodulin--The influence of the exchange of helical regions on calcium-binding affinity.

The four calcium-binding sites, called the helix-loop-helix, or the EF-hand motifs, of calmodulin differ in their ion-binding affinities; this has been thought to arise due to the variations in the sequences of the loop regions where the ion binds. We focus attention here on the role of the flanking helical regions on the calcium-binding affinities. Peptides were synthesized in a manner that simulates the E and F helical flanks of site 4 (the strongest calcium-binding site of the calmodulin) to sandwich the loop sequences of sites 1, 2, 3 and 4 so as to produce peptides named 414, 424, 434 and 444, as well as using the helical flanks of site 1 (the weakest site) to produce peptides 111, 121, 131 and 141. Calcium binding was monitored using the calcium-mimic dye Stains-all (4,4,4',5'-dibenzo-3,3'-diethyl-9-methyl-thiacarbocyanine bromide). Binding abilities were seen to increase several-fold when the E and F helices of site 1 were replaced by those of site 4 (i.e., 111-414). In contrast, the intensity of circular dichroism induced in the absorption bands of the bound achiral dye decreased significantly when the helical flanks of site 4 were replaced with those of site 1 (i.e., 444-141). The helical flanks of site 4 impart greater binding ability to a given loop region, while the helical flanks of site 1 tend to weaken it.