Role of four conserved aspartic acid residues of EF-loops in the metal ion binding and in the self-assembly of ciliate <Emphasis Type="Italic">Euplotes octocarinatus</Emphasis> centrin

Ciliate Euplotes octocarinatus centrin (EoCen) is an EF-hand calcium-binding protein closely related to the prototypical calcium sensor protein calmodulin. Four mutants (D37K, D73K, D110K and D146K) were created firstly to elucidate the importance of the first aspartic acid residues (Asp37, Asp73, Asp110 and Asp146) in the beginning of the four EF-loops of EoCen. Aromatic-sensitized Tb³⁺ fluorescence indicates that the aspartic acid residues are very important for the metal-binding of EoCen, except for Asp73 (in EF-loop II). Resonance light scattering (RLS) measurements for different metal ions (Ca²⁺ and Tb³⁺) binding proteins suggest that the order of four conserved aspartic acid residues for contributing to the self-assembly of EoCen is Asp37 > Asp146 > Asp110 > Asp73. Cross-linking experiment also exhibits that Asp37 and Asp146 play critical role in the self-assembly of EoCen. Asp37, in site I, which is located in the N-terminal domain, plays the most important role in the metal ion-dependent self-assembly of EoCen, and there is cooperativity between N-terminal and C-terminal domain (especially the site IV). In addition, the dependence of Tb³⁺ induced self-assembly of EoCen and the mutants on various factors, including ionic strength and pH, were characterized using RLS. Finally, 2-p-toluidinylnaphthalene-6-sulfonate (TNS) binding, ionic strength and pH control experiments indicate that in the process of EoCen self-assembly, molecular interactions are mediated by both electrostatic and hydrophobic forces, and the hydrophobic interaction has the important status.     

Calcium-Binding Properties of the Mitochondrial Channel-Forming Hydrophobic Component

A hydrophobic, low-molecular weight component extracted from mitochondria forms aCa²⁺-activated ion channel in black-lipid membranes (Mironova et al., 1997). At pH 8.3–8.5, thecomponent has a high-affinity binding site for Ca²⁺ with a K_d of 8 × 10^–6 M, while at pH7.5 this K_d was decreased to 9 × 10^–5 M. B_max for the Ca²⁺-binding site did not changesignificantly with pH. In the range studied, 0.2 ± 0.06 mmol Ca²⁺/g component were boundor one calcium ion to eight molecules of the component. The Ca²⁺ binding was stronglydecreased by 50–100 mM Na⁺, but not by K⁺. Treatment of mitochondria withCaCl₂ priorto ethanolic extraction resulted in a high level of Ca²⁺-binding capacity of the partially purifiedcomponent. Cyclosporin A, a specific inhibitor of the mitochondrial permeability transition,when added to the mitochondrial suspension, decreased the Ca²⁺-binding activity of thepurified extract severalfold. The calcium-binding capability of the partially purified componentcorrelates with its calcium-channel activity. This indicates that the channel-forming componentmight be involved in the permeability transition that stimulates its formation.     

Δ 469 mutation in the type 3 repeat calcium binding domain of cartilage oligomeric matrix protein (COMP) disrupts calcium binding

Cartilage oligomeric matrix protein (COMP/TSP5), a large glycoprotein found in the territorial matrix surrounding chondrocytes, is the fifth member of the thrombospondin (TSP) gene family. While the function of COMP is unknown, its importance is underscored by the finding that mutations in the highly conserved type 3 repeat domain causes two skeletal dysplasias. Pseudoachondroplasia (PSACH) and Multiple Epiphyseal Dysplasia, Fairbanks type (EDM1). The type 3 repeats are highly conserved low-affinity Ca²⁺binding domains that are found in all TSP genes. This study was undertaken to determine the effects of mutations on calcium binding and structure of the type 3 repeat domains. Wild-type (WT) and Δ469 recombinant COMP (rCOMP) proteins containing the entire calcium-binding domain were expressed in E. coli and purified. Equilibrium dialysis demonstrated that WT bound 10–12 Ca²⁺ions/molecule while Δ469 bound approximately half the Ca²⁺ions. Circular dichroism (CD) spectrometry had striking spectral changes for the WT in response to increasing concentrations of Ca²⁺. These CD spectral changes were cooperative and reversible. In contrast, a large CD spectral change was not observed at any Ca²⁺concentration for Δ469. Moreover, both WT and Δ469 proteins produced similar CD spectral changes when titrated with Zn²⁺, Cu²⁺and Ni²⁺indicating that the Δ469 mutation specifically affects only calcium binding. These results suggest that the Δ469 mutation, in the type 3 repeat region, interferes with Ca²⁺binding and that filling of all Ca²⁺binding loops may be critical for correct COMP protein conformation.     

Extra EF Hand Unit (DX) Mediated Stabilization and Calcium Independency of α-Amylase

It is the common feature of α-amylases that calcium ion is required for their structural integrity and thermal stability. All amylases have at least one Ca²⁺ per molecule; therefore amino acids involved in calcium binding are specific and conserved. In this study, sequence analysis revealed the presence of EF-hand-like motif in calcium-binding loop of Bacillus megaterium WHO (BMW)-amylase that was previously isolated from BMW. The EF-hand motif and its variants (EF-hand-like motif) are the most common calcium-binding motifs found in a large number of protein families. To investigate the effect of calcium ion on the thermal stability and activity of BMW-amylase, we used site-directed mutagenesis to replace histidine 58 with Asp (D), Ile (I), Tyr (Y), Phe (F), and Arg (R) at the seventh position of EF-hand-like motif. Upon the addition of an extra DX unit to the calcium-binding loop in H58D variant, thermal stability, catalytic activity, and chelating power of the enzyme improved due to higher affinity toward calcium. H58D variant demonstrated calcium independency compared to the wild type and other created mutants. Conformational changes in the presence and absence of Ca²⁺ were monitored using fluorescence technique.     

Engineered glucose isomerase from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. SK is resistant to Ca<Superscript>2+</Superscript> inhibition and Co<Superscript>2+</Superscript> independent

The role of two amino acid residues linked to the two catalytic histidines His54 and His220 in kinetics and physicochemical properties of the Streptomyces sp. SK glucose isomerase (SKGI) was investigated by site-directed mutagenesis and molecular modeling. Two single mutations, F53L and G219D, and a double mutation F53L/G219D was introduced into the xylA SKGI gene. The F53L mutation increases the thermostability and the catalytic efficiency and also slightly shifts the optimum pH from 6.5 to 7, but displays a profile being similar to that of the wild-type enzyme concerning the effect of various metal ions. The G219D mutant is resistant to calcium inhibition retaining about 80% of its residual activity in 10 mM Ca²⁺ instead of 10% for the wild-type. This variant is activated by Mn²⁺ ions, but not Co²⁺, as seen for the wild-type enzyme. It does not require the latter for its thermostability, but has its half-life time displaced from 50 to 20 min at 85°C. The double mutation F53L/G219D restores the thermostability as seen for the wild-type enzyme while maintaining the resistance to the calcium inhibition. Molecular modeling suggests that the increase in thermostability is due to new hydrophobic interactions stabilizing α2 helix and that the resistance to calcium inhibition is a result of narrowing the binding site of catalytic ion.     

Functional importance of calcium binding sites in outer membrane phospholipase A

Outer membrane phospholipase A (OMPLA) is an integral membrane enzyme that hydrolyses phospholipids requiring Ca²⁺ as cofactor. In vitro studies have shown that OMPLA is only active as a dimer. The structures of monomeric and dimeric OMPLA provided possible clues to the activation process. In the inhibited dimeric species calcium ions are located at the dimer interface ideally suited to stabilise the oxyanion intermediates formed during catalysis. The side chain hydroxyl function of Ser152 is one of the ligands of this interfacial calcium. In the crystal structure of monomeric OMPLA the interfacial calcium site is lacking, but calcium was found to bind at a site involving the carboxylates of Asp149 and Asp184. In the current study the relevance of the identified calcium sites has been studied by site-directed mutagenesis. The Ser152Asn variant confirmed the importance of the interfacial calcium site for catalysis, and also demonstrated that this site is essentially involved in the dimerisation process. Replacements of the ligands in monomeric OMPLA, i.e. Asp149Asn, Asp149Ala and Asp184Asn, only showed minor effects on catalytic activity and dimerisation. A stronger effect observed for the variant Asp184Ala was explained by the proximity of Asp184 to the catalytically important Ser152 residue. We propose that Asp149 and Asp184 provide an electronegative funnel that may facilitate Ca²⁺ transfer to the interfacial calcium site.     

Effects of Metal Ions on Stability and Activity of Hyperthermophilic Pyrolysin and Further Stabilization of This Enzyme by Modification of a Ca2+-Binding Site

Pyrolysin is an extracellular subtilase produced by the marine hyperthermophilic archaeon Pyrococcus furiosus. This enzyme functions at high temperatures in seawater, but little is known about the effects of metal ions on the properties of pyrolysin. Here, we report that the supplementation of Na⁺, Ca²⁺, or Mg²⁺ salts at concentrations similar to those in seawater destabilizes recombinant pyrolysin but leads to an increase in enzyme activity. The destabilizing effect of metal ions on pyrolysin appears to be related to the disturbance of surface electrostatic interactions of the enzyme. In addition, mutational analysis of two predicted high-affinity Ca²⁺-binding sites (Ca1 and Ca2) revealed that the binding of Ca²⁺ is important for the stabilization of this enzyme. Interestingly, Asn substitutions at residues Asp818 and Asp820 of the Ca2 site, which is located in the C-terminal extension of pyrolysin, resulted in improvements in both enzyme thermostability and activity without affecting Ca²⁺-binding affinity. These effects were most likely due to the elimination of unfavorable electrostatic repulsion at the Ca2 site. Together, these results suggest that metal ions play important roles in modulating the stability and activity of pyrolysin.     

The role of the Ca2+ binding ligand Asn879 in the function of the plasma membrane Ca2+ pump

Asn879 in the transmembrane segment M6 of the plasma membrane Ca²⁺ pump (PMCA human isoform 4xb) has been proposed to coordinate Ca²⁺ at the transport site through its carboxylate. This idea agrees with the fact that this Asn is conserved in other Ca²⁺-ATPases but is replaced by Asp, Glu, and other residues in closely related 2P-type ATPases of different ionic specificity. Previous mutagenesis studies have shown that the substitution of Ala for Asn abolishes the activity of the enzyme (Adebayo et al., 1995; Guerini et al., 1996). We have constructed a mutant PMCA in which the Asn879 was substituted by Asp. The mutant protein was expressed in Saccharomyces cerevisiae, solubilized and purified by calmodulin affinity chromatography. The Asn879Asp PMCA mutant exhibited about 30% of the wild type Ca²⁺-dependent ATPase activity and only a minor reduction of the apparent affinity for Ca²⁺. The decrease in the Ca²⁺-ATPase of the mutant enzyme was in parallel with the reduction in the amount of phosphoenzyme formed from Ca²⁺ plus ATP. Noteworthy, the mutation nearly eliminated the ability of the enzyme to hydrolyze pNPP which is maximal in the absence of Ca²⁺ revealing a major effect of the mutation on the Ca²⁺-independent reactions of the transport cycle. At a pH low enough to protonate the Asp carboxylate the pNPPase activity of Asn879Asp increased, suggesting that the binding of protons to Asn879 is essential for the activities catalyzed by E₂-like forms of the enzyme.     

Functional Roles of the Non-Catalytic Calcium-Binding Sites in the N-Terminal Domain of Human Peptidylarginine Deiminase 4

This study investigated the functional roles of the N-terminal Ca²⁺ ion-binding sites, in terms of enzyme catalysis and stability, of peptidylarginine deiminase 4 (PAD4). Amino acid residues located in the N-terminal Ca²⁺-binding site of PAD4 were mutated to disrupt the binding of Ca²⁺ ions. Kinetic data suggest that Asp155, Asp157 and Asp179, which directly coordinate Ca3 and Ca4, are essential for catalysis in PAD4. For D155A, D157A and D179A, the k _cat/K _m,BAEE values were 0.02, 0.63 and 0.01 s⁻¹mM⁻¹ (20.8 s⁻¹mM⁻¹ for WT), respectively. Asn153 and Asp176 are directly coordinated with Ca3 and indirectly coordinated with Ca5 via a water molecule. However, N153A displayed low enzymatic activity with a k _cat value of 0.3 s⁻¹ (13.3 s⁻¹ for wild-type), whereas D176A retained some catalytic power with a k _cat of 9.7 s⁻¹. Asp168 is the direct ligand for Ca5, and Ca5 coordination by Glu252 is mediated by two water molecules. However, mutation of these two residues to Ala did not cause a reduction in the k _cat/K _m,BAEE values, which indicates that the binding of Ca5 may not be required for PAD4 enzymatic activity. The possible conformational changes of these PAD4 mutants were examined. Thermal stability analysis of the PAD4 mutants in the absence or presence of Ca²⁺ indicated that the conformational stability of the enzyme is highly dependent on Ca²⁺ ions. In addition, the results of urea-induced denaturation for the N153, D155, D157 and D179 series mutants further suggest that the binding of Ca²⁺ ions in the N-terminal Ca²⁺-binding site stabilizes the overall conformational stability of PAD4. Therefore, our data strongly suggest that the N-terminal Ca²⁺ ions play critical roles in the full activation of the PAD4 enzyme.     

Crystal Structures of A. acidocaldarius Endoglucanase Cel9A in Complex with Cello-Oligosaccharides: Strong − 1 and − 2 Subsites Mimic Cellobiohydrolase Activity

Alicyclobacillus acidocaldarius endoglucanase Cel9A (AaCel9A) is an inverting glycoside hydrolase with β-1,4-glucanase activity on soluble polymeric substrates. Here, we report three X-ray structures of AaCel9A: a ligand-free structure at 1.8 Å resolution and two complexes at 2.66 and 2.1 Å resolution, respectively, with cellobiose obtained by co-crystallization and with cellotetraose obtained by the soaking method. AaCel9A forms an (α/α)₆-barrel like other glycoside hydrolase family 9 enzymes. When cellobiose is used as a ligand, three glucosyl binding subsites are occupied, including two on the glycone side, while with cellotetraose as a ligand, five subsites, including four on the glycone side, are occupied. A structural comparison showed no conformational rearrangement of AaCel9A upon ligand binding. The structural analysis demonstrates that of the four minus subsites identified, subsites − 1 and − 2 show the strongest interaction with bound glucose. In conjunction with the open active-site cleft of AaCel9A, this is able to reconcile the previously observed cleavage of short-chain oligosaccharides with cellobiose as main product with the endo mode of action on larger polysaccharides.     

Exact Stochastic Simulation of a Calcium Microdomain Reveals the Impact of Ca2+ Fluctuations on IP3R Gating

In this study, we numerically analyzed the nonlinear Ca²⁺-dependent gating dynamics of a single, nonconducting inositol 1,4,5-trisphosphate receptor (IP₃R) channel, using an exact and fully stochastic simulation algorithm that includes channel gating, Ca²⁺ buffering, and Ca²⁺ diffusion. The IP₃R is a ubiquitous intracellular Ca²⁺ release channel that plays an important role in the formation of complex spatiotemporal Ca²⁺ signals such as waves and oscillations. Dynamic subfemtoliter Ca²⁺ microdomains reveal low copy numbers of Ca²⁺ ions, buffer molecules, and IP₃Rs, and stochastic fluctuations arising from molecular interactions and diffusion do not average out. In contrast to models treating calcium dynamics deterministically, the stochastic approach accounts for this molecular noise. We varied Ca²⁺ diffusion coefficients and buffer reaction rates to tune the autocorrelation properties of Ca²⁺ noise and found a distinct relation between the autocorrelation time τ_ac, the mean channel open and close times, and the resulting IP₃R open probability P_O. We observed an increased P_O for shorter noise autocorrelation times, caused by increasing channel open times and decreasing close times. In a pure diffusion model the effects become apparent at elevated calcium concentrations, e.g., at [Ca²⁺] = 25 μM, τ_ac = 0.082 ms, the IP₃R open probability increased by ≈20% and mean open times increased by ≈4 ms, compared to a zero noise model. We identified the inactivating Ca²⁺ binding site of IP₃R subunits as the primarily noise-susceptible element of the De Young and Keizer model. Short Ca²⁺ noise autocorrelation times decrease the probability of Ca²⁺ association and consequently increase IP₃R activity. These results suggest a functional role of local calcium noise properties on calcium-regulated target molecules such as the ubiquitous IP₃R. This finding may stimulate novel experimental approaches analyzing the role of calcium noise properties on microdomain behavior.     

Differential contribution of EF-hands to the Ca²⁺-dependent activation in the plant two-pore channel TPC1

Two‐pore channels (TPC) have been established as components of calcium signalling networks in plants and animals. In plants, TPC1 in the vacuolar membrane is gated open upon binding of calcium in a voltage‐dependent manner. Here, we analyzed the molecular mechanism of the Ca²⁺‐dependent activity of TPC1 from Arabidopsis thaliana, using site‐directed mutagenesis of its two canonical EF‐hands. Wild‐type TPC1 and TPC1‐D335A with a mutated first Ca²⁺ ligand in EF‐hand 1 produced channels that retained their voltage‐ and Ca²⁺‐dependent gating characteristics, but were less sensitive at Ca²⁺ concentrations <200 μm . Additional mutation of the first Ca²⁺ ligand in EF‐hand 2 resulted in silent TPC1‐D335A/D376A channels. Similarly, the single mutant TPC1‐D376A could not be activated up to 1 mm Ca²⁺, indicating that the second EF‐hand is essential for the Ca²⁺‐dependent channel gating. Molecular modeling suggests that EF‐hand 1 displays a low‐affinity Ca²⁺/Mg²⁺‐binding site, while EF‐hand 2 represents a high‐affinity Ca²⁺‐binding site. Together, our data prove that EF‐hand 2 is responsible for the Ca²⁺‐receptor characteristics of TPC1, while EF‐hand 1 is a structural site required to enable channel responses at physiological changes in Ca²⁺ concentration.     

Long-Timescale Molecular Dynamics Simulations Elucidate the Dynamics and Kinetics of Exposure of the Hydrophobic Patch in Troponin C

Troponin (Tn) is an important regulatory protein in the thin-filament complex of cardiomyocytes. Calcium binding to the troponin C (TnC) subunit causes a change in its dynamics that leads to the transient opening of a hydrophobic patch on TnC’s surface, to which a helix of another subunit, troponin I (TnI), binds. This process initiates contraction, making it an important target for studies investigating the detailed molecular processes that underlie contraction. Here we use microsecond-timescale Anton molecular dynamics simulations to investigate the dynamics and kinetics of the opening transition of the TnC hydrophobic patch. Free-energy differences for opening are calculated for wild-type Ca²⁺-bound TnC (∼8 kcal/mol), V44Q Ca²⁺-bound TnC (3.2 kcal/mol), E40A Ca²⁺-bound TnC (∼12 kcal/mol), and wild-type apo TnC (∼20 kcal/mol). These results suggest that the mutations have a profound impact on the frequency with which the hydrophobic patch presents to TnI. In addition, these simulations corroborate that cardiac wild-type TnC does not open on timescales relevant to contraction without calcium being bound.     

Studies on Cellulose Hydrolysis by Acetivibrio cellulolyticus

Acetivibrio cellulolyticus extracellular cellulase extensively hydrolyzed crystalline celluloses such as Avicel (FMC Corp., Food and Pharmaceutical Products Div., Philadelphia, Pa.) but only if it was desalted and supplemented with Ca²⁺. The Ca²⁺ effect was one of increased enzyme stability in the presence of the ion. Although preincubation of the cellulase complex at 40°C for 5 h without added Ca²⁺ had a negligible effect on endoglucanase activity or on the subseqent hydrolysis of amorphous cellulose, the capacity of the enzyme to hydrolyze crystalline cellulose was almost completely lost. Adsorption studies showed that 90% of the Avicel-solubilizing component of the total enzyme preparation bound to 2% Avicel at 40°C. Under these conditions, only 15% of the endoglucanase and 25% of the protein present in the enzyme preparation adsorbed to the substrate. The protein profile of the bound enzyme, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was complex and distinctly different from the profile observed for total cellulase preparations. The specific activity of A. cellulolyticus cellulase with respect to Avicel hydrolysis was compared with that of commercially available Trichoderma reesei cellulase.     

Neuronal Calcium Sensor-1 Binds the D2 Dopamine Receptor and G-protein-coupled Receptor Kinase 1 (GRK1) Peptides Using Different Modes of Interactions

Neuronal calcium sensor-1 (NCS-1) is the primordial member of the neuronal calcium sensor family of EF-hand Ca²⁺-binding proteins. It interacts with both the G-protein-coupled receptor (GPCR) dopamine D2 receptor (D2R), regulating its internalization and surface expression, and the cognate kinases GRK1 and GRK2. Determination of the crystal structures of Ca²⁺/NCS-1 alone and in complex with peptides derived from D2R and GRK1 reveals that the differential recognition is facilitated by the conformational flexibility of the C-lobe-binding site. We find that two copies of the D2R peptide bind within the hydrophobic crevice on Ca²⁺/NCS-1, but only one copy of the GRK1 peptide binds. The different binding modes are made possible by the C-lobe-binding site of NCS-1, which adopts alternative conformations in each complex. C-terminal residues Ser-178–Val-190 act in concert with the flexible EF3/EF4 loop region to effectively form different peptide-binding sites. In the Ca²⁺/NCS-1·D2R peptide complex, the C-terminal region adopts a 3₁₀ helix-turn-3₁₀ helix, whereas in the GRK1 peptide complex it forms an α-helix. Removal of Ser-178–Val-190 generated a C-terminal truncation mutant that formed a dimer, indicating that the NCS-1 C-terminal region prevents NCS-1 oligomerization. We propose that the flexible nature of the C-terminal region is essential to allow it to modulate its protein-binding sites and adapt its conformation to accommodate both ligands. This appears to be driven by the variability of the conformation of the C-lobe-binding site, which has ramifications for the target specificity and diversity of NCS-1.     

Cloning,expression of b-1,3-1,4 glucanase from Bacillus subtilis SU40 and the effect of calcium ion on the stability of recombinant enzyme: in vitro and in silico analysis

A new glucanolytic bacterial strain, SU40 was isolated, and identified as Bacillus subtilis on the basis of 16S rRNA sequence homology and phylogenetic tree analysis. The gene encoding β-1,3-1,4-glucanase was delineated, cloned into pET 28a+ vector and heterologously overexpressed in Escherichia coli BL21(DE3). The purified recombinant enzyme was about 24 kDa. The enzyme exhibited maximum activity (36.84 U/ml) at 60°C, pH 8.0 and maintained 54% activity at 80°C after incubation for 60 min. The enzyme showed activity against β-glucan, lichenan, and xylan. Amino acid sequence shared a conserved motif EIDIEF. The predicted three-dimensional homology model of the enzyme showed the presence of catalytic residues Glu105, Glu109 and Asp107, single disulphide bridge between Cys32 and Cys61 and three calcium binding site residues Pro9, Gly45 and Asp207. Presence of calcium ion improves the thermal stability of SU40 β-1,3-1,4-glucanase. Molecular dynamics simulation studies revealed that the absence of calcium ion fluctuate the active site residues which are responsible for thermostability. The high catalytic activity and its stability to temperature, pH and metal ions indicated that the enzyme β-1,3-1,4-glucanase by B. subtilis SU40 is a good candidate for biotechnological applications.     

Molecular Basis of S100A1 Activation at Saturating and Subsaturating Calcium Concentrations

The S100A1 protein mediates a wide variety of physiological processes through its binding of calcium (Ca²⁺) and endogenous target proteins. S100A1 presents two Ca²⁺-binding domains: a high-affinity “canonical” EF (cEF) hand and a low-affinity “pseudo” EF (pEF) hand. Accumulating evidence suggests that both Ca²⁺-binding sites must be saturated to stabilize an open state conducive to peptide recognition, yet the pEF hand’s low affinity limits Ca²⁺ binding at normal physiological concentrations. To understand the molecular basis of Ca²⁺ binding and open-state stabilization, we performed 100 ns molecular dynamics simulations of S100A1 in the apo/holo (Ca²⁺-free/bound) states and a half-saturated state, for which only the cEF sites are Ca²⁺-bound. Our simulations indicate that the pattern of oxygen coordination about Ca²⁺ in the cEF relative to the pEF site contributes to the former’s higher affinity, whereas Ca²⁺ binding strongly reshapes the protein’s conformational dynamics by disrupting β-sheet coupling between EF hands. Moreover, modeling of the half-saturated configuration suggests that the open state is unstable and reverts toward a closed state in the absence of the pEF Ca²⁺ ion. These findings indicate that Ca²⁺ binding at the cEF site alone is insufficient to stabilize opening; thus, posttranslational modification of the protein may be required for target peptide binding at subsaturating intracellular Ca²⁺ levels.     

The contribution of the cell wall to a transmembrane calcium gradient could play a key role in Bacillus subtilis protein secretion

A weak Ca²⁺-binding site (K_a= 0.8× 10³ M^?1, at pH7) was identified in the mature part of levansucrase. An amino acid substitution (Thr-236 →lle) in this site alters simultaneously the affinity for calcium, the folding transition and the efficiency of the secretion process of levansucrase. Moreover, the ability of the Bacillus subtilis cell wall to concentrate calcium ions present in the culture medium was studied. We confirm the results of Beveridge and Murray who showed that the concentration factor is about 100 to 120 times. This property preserves a high concentration of Ca²⁺ (>2 mM) on the external side of the cytoplasmic membrane, even in the absence of further Ca2+ supplementation in the growth medium. Such local conditions allow the spontaneous unfolding folding transition of levansucrase en route for secretion. Since several exocellular proteins of B. subtilis are calcium-binding proteins, we propose that the high concentration of calcium ion in the microenvironment of the cell wall may play a key role in the ultimate step of their secretion process.     

Expression and characterization of a novel mesophilic protease from metagenomic library derived from Antarctic coastal sediment

A metagenomic cosmid library was constructed, in which the insert DNA was derived from the coastal sediment near Antarctic China Zhongshan Station. One clone (ACPRO001) expressing protease activity was isolated from the library using milk agar plates. Sequencing of the clone revealed a novel protease gene. The amino acid sequence comparison and phylogenetic analysis indicated that it could be classified as a subtilisin-like serine protease, though the highly conserved residue Asp was replaced by Ala. The ACPRO001 protease gene was expressed in pET-His and purified for characterization. The optimal temperature and pH for the activity of the ACPRO001 protease were 60°C and pH 9.0, respectively. The enzyme retained about 73% of residual activity after 2 h incubation at 50°C in the presence of Ca²⁺. The presence of Ca²⁺ increased the thermostability of ACPRO001 protease obviously. The enzymatic activity was inhibited by 1 mM phenylmethyl sulfonylfluoride (PMSF) and hydrochloride 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), indicating that it was a serine protease.     

The relationship between vitamin D-stimulated calcium transport and intestinal calcium-binding protein in the chicken

1. The rapid stimulation of intestinal Ca²⁺ transport observed in vitamin D-deficient chicks after receiving 1,25-dihydroxycholecalciferol has necessitated a re-evaluation of the correlation hitherto observed between this stimulation and the induction of calcium-binding protein synthesis. By 1h after a dose of 125ng of 1,25-dihydroxycholecalciferol, Ca²⁺ transport is increased. This is at least 2h before calcium-binding protein can be detected immunologically and 1h before synthesis of the protein begins on polyribosomes, and thus the hormone stimulates Ca²⁺ transport before calcium-binding-protein biosynthesis is induced. 2. The maximum increase in Ca²⁺ transport observed after this dose of 1,25-dihydroxycholecalciferol (attained by 8h) is similar to that observed after 1.25–25μg of cholecalciferol, but the stimulation is only short-lived, in contrast with the effect observed after the vitamin. At later times after the hormone, however, when Ca²⁺ transport has declined to its basal rate, the cellular content of calcium-binding protein remains elevated. 3. Calcium-binding protein is synthesized on free rather than membrane-bound polyribosomes, which implies that it is an intracellular protein. 4. Rachitic chicks require the presence of dietary calcium for maximum stimulation of calcium-binding protein production by cholecalciferol. 5. These results suggest that calcium-binding protein is an intracellular protein, and that its synthesis may be a consequence of the raised intracellular calcium content of the intestinal epithelial cells resulting from 1,25-dihydroxycholecalciferol-stimulated Ca²⁺ transport. We propose that calcium-binding-protein synthesis is necessary for maintaining the stimulated rate of Ca²⁺ transport, which is initiated by other factors.