Effect of metal ions on the activity of green crab (Scylla serrata) alkaline phosphatase

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. The present paper deals with the study of the effect of some kinds of metal ions on the enzyme. The positive monovalent alkali metal ions (Li(+), Na(+) and K(+)) have no effect on the enzyme; positive bivalent alkaline-earth metal ions (Mg(2+), Ca(2+) and Ba(2+)) and transition metal ions (Mn(2+), Co(2+), Ni(2+) and Cd(2+)) activate the enzyme; heavy metal ions (Hg(2+), Ag(+), Bi(2+), Cu(2+) and Zn(2+)) inhibit the enzyme. The activation of magnesium ion on the enzyme appears to be a partial noncompetitive type. The kinetic model has been set up and a new plot to determine the activation constant of Mg(2+) was put forward. From the plot, we can easily determine the activation constant (K(a)) value and the activation ratio of Mg(2+) on the enzyme. The inhibition effects of Cu(2+) and Hg(2+) on the enzyme are of noncompetitive type. The inhibition constants have been determined. The inhibition effect of Hg(2+) is stronger than that of Cu(2+).     

Biophysical and structural studies of the human calcium- and integrin-binding protein family: understanding their functional similarities and differences

The human calcium- and integrin-binding protein 1 (CIB1) plays important roles in various cellular functions. In this study, three other members of this protein family (CIB2-4: CIB2, CIB3, and CIB4) were purified and subsequently characterized using biophysical and structural approaches. As expected from sequence alignments, CIB2-4 were shown to bind calcium (Ca(2+)) and magnesium (Mg(2+)) ions. Binding of Ca(2+) or Mg(2+) ions changes the secondary structure of CIB2-4 and the exposure of hydrophobic surface area. Ca(2+) and Mg(2+) ions also stabilize the tertiary structures for CIB2 and CIB3. Through in vitro binding experiments, we show that CIB2 can interact with the integrin αIIb cytoplasmic domain and the integrin α7b membrane-proximal fragment. Fluorescence experiments using a 7-azatryptophan labeled peptide demonstrate that CIB2, CIB3, and CIB4 are binding partners for the integrin αIIb subunit, which suggests that they are potentially involved in regulating integrin αIIb subunit activation. The distinct responses of αIIb to the different CIB3 and CIB4 metal (Ca(2+) and Mg(2+)) binding states imply a potential connection between the calcium and integrin signaling pathways.     

Kinetics of conformational changes induced by the binding of various metal ions to bovine alpha-lactalbumin.

The kinetic effects of the binding of various metal ions (Ca(2+), Cd(2+), Co(2+), Mg(2+), Mn(2+), Sr(2+) and Zn(2+)) to apo bovine alpha-lactalbumin has been monitored by means of stopped-flow fluorescence spectroscopy. Our results show that the measured rate constant for the binding of metal ions to the Ca(2+)-site increases with increasing binding constant. This is, however, not the case for metal ions binding to the Zn(2+)-site. The binding experiments performed at different temperatures allowed us to calculate the activation energy for the transition from the metal-free to the metal-loaded state of the protein. These values do not depend on the nature of the metal ion but are correlated with the type of binding site. As a result, we were able to demonstrate that Mg(2+), a metal ion which was thought to bind to the Ca(2+)-site, shows the same binding characteristics as Co(2+) and Zn(2+) and therefore most likely interacts with the residues belonging to the Zn(2+)-binding site.     

Interaction of group IA phospholipase A2 with metal ions and phospholipid vesicles probed with deuterium exchange mass spectrometry

Deuterium exchange mass spectrometric evaluation of the cobra venom (Naja naja naja) group IA phospholipase A 2 (GIA PLA 2) was carried out in the presence of metal ions Ca (2+) and Ba (2+) and phospholipid vesicles. Novel conditions for digesting highly disulfide bonded proteins and a methodology for studying protein-lipid interactions using deuterium exchange have been developed. The enzyme exhibits unexpectedly slow rates of exchange in the two large alpha-helices of residues 43-53 and 89-101, which suggests that these alpha-helices are highly rigidified by the four disulfide bonds in this region. The binding of Ca (2+) or Ba (2+) ions decreased the deuterium exchange rates for five regions of the protein (residues 24-27, 29-40, 43-53, 103-110, and 111-114). The magnitude of the changes was the same for both ions with the exception of regions of residues 24-27 and 103-110 which showed greater changes for Ca (2+). The crystal structure of the N. naja naja GIA PLA 2 contains a single Ca (2+) bound in the catalytic site, but the crystal structures of related PLA 2s contain a second Ca (2+) binding site. The deuterium exchange studies reported here clearly show that in solution the GIA PLA 2 does in fact bind two Ca (2+) ions. With dimyristoylphosphatidylcholine (DMPC) phospholipid vesicles with 100 microM Ca (2+) present at 0 degrees C, significant areas on the i-face of the enzyme showed decreases in the rate of exchange. These areas included regions of residues 3-8, 18-21, and 56-64 which include Tyr-3, Trp-61, Tyr-63, and Phe-64 proposed to penetrate the membrane surface. These regions also contained Phe-5 and Trp-19, proposed to bind the fatty acyl tails of substrate.     

NADH fluorescence lifetime analysis of the effect of magnesium ions on ALDH2

Aldehyde dehydrogenase 2 (ALDH2) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg(2+) ions influence ALDH2 activity in part by increasing NADH binding affinity. Traditional fluorescence measurements monitor the blue shift of the NADH fluorescence spectrum to study ALDH2-NADH interactions. By using time-resolved fluorescence spectroscopy, we have resolved the fluorescent lifetimes (τ) of free NADH (τ=0.4 ns) and bound NADH (τ=6.0 ns). We used this technique to investigate the effects of Mg(2+) on the ALDH2-NADH binding characteristics and enzyme catalysis. From the resolved free and bound NADH fluorescence signatures, the K(D) for NADH with ALDH2 ranged from 468 μM to 12 μM for Mg(2+) ion concentrations of 20 to 6000 μM, respectively. The rate constant for dissociation of the enzyme-NADH complex ranged from 0.4s(-1) (6000 μM Mg(2+)) to 8.3s(-1) (0 μM Mg(2+)) as determined by addition of excess NAD(+) to prevent re-association of NADH and resolving the real-time NADH fluorescence signal. The apparent NADH association/re-association rate constants were approximately 0.04 μM(-1)s(-1) over the entire Mg(2+) ion concentration range and demonstrate that Mg(2+) ions slow the release of NADH from the enzyme rather than promoting its re-association. We applied NADH fluorescence lifetime analysis to the study of NADH binding during enzyme catalysis. Our fluorescence lifetime analysis confirmed complex behavior of the enzyme activity as a function of Mg(2+) concentration. Importantly, we observed no pre-steady state burst of NADH formation. Furthermore, we observed distinct fluorescence signatures from multiple ALDH2-NADH complexes corresponding to free NADH, enzyme-bound NADH, and, potentially, an abortive NADH-enzyme-propanal complex (τ=11.2 ns).     

Modulation of cardiac ryanodine receptor channels by alkaline earth cations

Cardiac ryanodine receptor (RyR2) function is modulated by Ca(2+) and Mg(2+). To better characterize Ca(2+) and Mg(2+) binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M(2+): Mg(2+), Ca(2+), Sr(2+), Ba(2+)) were studied on RyR2 from pig ventricle reconstituted in bilayers. RyR2 were activated by M(2+) binding to high affinity activating sites at the cytosolic channel surface, specific for Ca(2+) or Sr(2+). This activation was interfered by Mg(2+) and Ba(2+) acting at low affinity M(2+)-unspecific binding sites. When testing the effects of luminal M(2+) as current carriers, all M(2+) increased maximal RyR2 open probability (compared to Cs(+)), suggesting the existence of low affinity activating M(2+)-unspecific sites at the luminal surface. Responses to M(2+) vary from channel to channel (heterogeneity). However, with luminal Ba(2+)or Mg(2+), RyR2 were less sensitive to cytosolic Ca(2+) and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca(2+)or Sr(2+)). Kinetics of RyR2 with mixtures of luminal Ba(2+)/Ca(2+) and additive action of luminal plus cytosolic Ba(2+) or Mg(2+) suggest luminal M(2+) differentially act on luminal sites rather than accessing cytosolic sites through the pore. This suggests the presence of additional luminal activating Ca(2+)/Sr(2+)-specific sites, which stabilize high P(o) mode (less voltage-dependent) and increase RyR2 sensitivity to cytosolic Ca(2+) activation. In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M(2+) binding sites (specific for Ca(2+) and unspecific for Ca(2+)/Mg(2+)) that dynamically modulate channel activity and gating status, depending on SR voltage.     

Metal binding to ligands: cadmium complexes with glutathione revisited

We studied the interaction of gamma-L-glutamyl-L-cysteinyl-glycine (glutathione, GSH) with cadmium ions (Cd(2+)) by first performing classical potentiometric pH titration measurements and then turning to additional spectroscopic methods. To estimate the residual concentrations of free cadmium, we studied the competition of glutathione with a Cd(2+)-sensitive dye, either an absorbing dye (murexide) or a fluorescent one (FluoZin-1), and consistent results were obtained with the two dyes. In KCl-containing Tes, Mops, or Tris buffer at pH 7.0 to 7.1 and 37 degrees C (and at a total Cd(2+) concentration of 0.01 mM), results suggest that free cadmium concentration is halved when the concentration of glutathione is approximately 0.05 mM; this mainly reflects the combined apparent dissociation constant for the Cd(glutathione) 1:1 complex under these conditions. To identify the other complexes formed, we used far-UV spectroscopy of the ligand-to-metal charge transfer absorption bands. The Cd(glutathione)(2) 1:2 complex predominated over the 1:1 complex only at high millimolar concentrations of total glutathione and not at low submillimolar concentrations of total glutathione. The apparent conditional constants derived from these spectroscopy results made it possible to discriminate between sets of absolute constants that would otherwise have simulated the pH titration data similarly well in this complicated system. Related experiments showed that although the Cl(-) ions in our media competed (modestly) with glutathione for binding to Cd(2+), the buffers we had chosen did not bind Cd(2+) significantly under our conditions. Our experiments also revealed that Cd(2+) may be adsorbed onto quartz or glass vessel walls, reducing the accuracy of theoretical predictions of the concentrations of species in solution. Lastly, the experiments confirmed the rapid kinetics of formation and dissociation of the UV-absorbing Cd(glutathione)(2) 1:2 complexes. The methods described here may be useful for biochemists needing to determine conditional binding constants for charge transfer metal-ligand complexes under their own conditions.     

Kinetic analysis of the interactions between troponin C (TnC) and troponin I (TnI) binding peptides: evidence for separate binding sites for the 'structural' N-terminus and the 'regulatory' C-terminus of TnI on TnC

The Ca(2+)/Mg(2+)-dependent interactions between TnC and TnI play a critical role in regulating the 'on' and 'off' states of muscle contraction as well as maintaining the structural integrity of the troponin complex in the off state. In the present study, we have investigated the binding interactions between the N-terminus of TnI (residues 1-40 of skeletal TnI) and skeletal TnC in the presence of Ca(2+) ions, Mg(2+) ions and in the presence of the C-terminal regulatory region peptides: TnI(96-115), TnI(96-131) and TnI(96-139). Our results show the N-terminus of TnI can bind to TnC with high affinity in the presence of Ca(2+) or Mg(2+) ions with apparent equilibrium dissociation constants of K(d(Ca(2+) ) ) = 48 nM and K(d(Mg(2+) ) ) = 29 nM. The apparent association and dissociation rate constants for the interactions were, k(on) = 4.8 x 10(5) M (-1) s(-1), 3.4 x 10(5) M (-1) s(-1) and k(off) = 2.3 x 10(-2) s(-1), 1.0 x 10(-2) s(-1) for TnC(Ca(2+)) and TnC(Mg(2+)) states, respectively. Competition studies between each of the TnI regions and TnC showed that both TnI regions can bind simultaneously to TnC while native gel electrophoresis and SEC confirmed the formation of stable ternary complexes between TnI(96-139) (or TnI(96-131)) and TnC-TnI(1-40). Further analysis of the binding interactions in the ternary complex showed the binding of the TnI regulatory region to TnC was critically dependent upon the presence of both TnC binding sites (i.e. TnI(96-115) and TnI(116-131)) and the presence of Ca(2+). Furthermore, the presence of TnI(1-40) slightly weakened the affinity of the regulatory peptides for TnC. Taken together, these results support the model for TnI-TnC interaction where the N-terminus of TnI remains bound to the C-domain of TnC in the presence of high and low Ca(2+) levels while the TnI regulatory region (residues 96-139) switches in its binding interactions between the actin-tropomyosin thin filament and its own sites on the N- and C-domain of TnC at high Ca(2+) levels, thus regulating muscle contraction.     

Ion interactions in the high-affinity binding locus of a voltage-gated Ca(2+) channel

The selectivity filter of voltage-gated Ca(2+) channels is in part composed of four Glu residues, termed the EEEE locus. Ion selectivity in Ca(2+) channels is based on interactions between permeant ions and the EEEE locus: in a mixture of ions, all of which can pass through the pore when present alone, those ions that bind weakly are impermeant, those that bind more strongly are permeant, and those that bind more strongly yet act as pore blockers as a consequence of their low rate of unbinding from the EEEE locus. Thus, competition among ion species is a determining feature of selectivity filter function in Ca(2+) channels. Previous work has shown that Asp and Ala substitutions in the EEEE locus reduce ion selectivity by weakening ion binding affinity. Here we describe for wild-type and EEEE locus mutants an analysis at the single channel level of competition between Cd(2+), which binds very tightly within the EEEE locus, and Ba(2+) or Li(+), which bind less tightly and hence exhibit high flux rates: Cd(2+) binds to the EEEE locus approximately 10(4)x more tightly than does Ba(2+), and approximately 10(8)x more tightly than does Li(+). For wild-type channels, Cd(2+) entry into the EEEE locus was 400x faster when Li(+) rather than Ba(2+) was the current carrier, reflecting the large difference between Ba(2+) and Li(+) in affinity for the EEEE locus. For the substitution mutants, analysis of Cd(2+) block kinetics shows that their weakened ion binding affinity can result from either a reduction in blocker on rate or an enhancement of blocker off rate. Which of these rate effects underlay weakened binding was not specified by the nature of the mutation (Asp vs. Ala), but was instead determined by the valence and affinity of the current-carrying ion (Ba(2+) vs. Li(+)). The dependence of Cd(2+) block kinetics upon properties of the current-carrying ion can be understood by considering the number of EEEE locus oxygen atoms available to interact with the different ion pairs.     

Evidence for a deep pore activation gate in small conductance Ca2+-activated K+ channels

Small conductance calcium-gated potassium (SK) channels share an overall topology with voltage-gated potassium (K(v)) channels, but are distinct in that they are gated solely by calcium (Ca(2+)), not voltage. For K(v) channels there is strong evidence for an activation gate at the intracellular end of the pore, which was not revealed by substituted cysteine accessibility of the homologous region in SK2 channels. In this study, the divalent ions cadmium (Cd(2+)) and barium (Ba(2+)), and 2-aminoethyl methanethiosulfonate (MTSEA) were used to probe three sites in the SK2 channel pore, each intracellular to (on the selectivity filter side of) the region that forms the intracellular activation gate of voltage-gated ion channels. We report that Cd(2+) applied to the intracellular side of the membrane can modify a cysteine introduced to a site (V391C) just intracellular to the putative activation gate whether channels are open or closed. Similarly, MTSEA applied to the intracellular side of the membrane can access a cysteine residue (A384C) that, based on homology to potassium (K) channel crystal structures (i.e., the KcsA/MthK model), resides one amino acid intracellular to the glycine gating hinge. Cd(2+) and MTSEA modify with similar rates whether the channels are open or closed. In contrast, Ba(2+) applied to the intracellular side of the membrane, which is believed to block at the intracellular end of the selectivity filter, blocks open but not closed channels when applied to the cytoplasmic face of rSK2 channels. Moreover, Ba(2+) is trapped in SK2 channels when applied to open channels that are subsequently closed. Ba(2+) pre-block slows MTSEA modification of A384C in open but not in closed (Ba(2+)-trapped) channels. The findings suggest that the SK channel activation gate resides deep in the vestibule of the channel, perhaps in the selectivity filter itself.     

Biochemical analysis of the interaction of calcium with toposome: a major protein component of the sea urchin egg and embryo

We have investigated the biochemical and functional properties of toposome, a major protein component of sea urchin eggs and embryos. Atomic force microscopy was utilized to demonstrate that a Ca(2+)-driven change in secondary structure facilitated toposome binding to a lipid bilayer. Thermal denaturation studies showed that toposome was dependent upon calcium in a manner paralleling the effect of this cation on secondary and tertiary structure. The calcium-induced, secondary, and tertiary structural changes had no effect on the chymotryptic cleavage pattern. However, the digestion pattern of toposome bound to phosphatidyl serine liposomes did vary as a function of calcium concentration. We also investigated the interaction of this protein with various metal ions. Calcium, Mg(2+), Ba(2+), Cd(2+), Mn(2+), and Fe(3+) all bound to toposome. In addition, Cd(2+) and Mn(2+) displaced Ca(2+), prebound to toposome, while Mg(2+), Ba(2+), and Fe(3+) had no effect. Collectively, these results further enhance our understanding of the role of Ca(2+) in modulating the biological activity of toposome.     

Identification of the magnesium ion binding site in the catalytic center of Escherichia coli primase by iron cleavage

Magnesium is essential for the catalysis reaction of Escherichia coli primase, the enzyme synthesizing primer RNA chains for initiation of DNA replication. To map the Mg(2+) binding site in the catalytic center of primase, we have employed the iron cleavage method in which the native bound Mg(2+) ions were replaced with Fe(2+) ions and the protein was then cleaved in the vicinity of the metal binding site by adding DTT which generated free hydroxyl radicals from the bound iron. Three Fe(2+) cleavages were generated at sites designated I, II, and III. Adding Mg(2+) or Mn(2+) ions to the reaction strongly inhibited Fe(2+) cleavage; however, adding Ca(2+) or Ba(2+) ions had much less effect. Mapping by chemical cleavage and subsequent site-directed mutagensis demonstrated that three acidic residues, Asp345 and Asp347 of a conserved DPD sequence and Asp269 of a conserved EGYMD sequence, were the amino acid residues that chelated Mg(2+) ions in the catalytic center of primase. Cleavage data suggested that binding to D345 is significantly stronger than to D347 and somewhat stronger than to D269.     

Binding of divalent metal ions to calcium-free peroxidase: thermodynamic and kinetic studies

Thermodynamics of binding of divalent metal ions including Ca(2+) , Mg(2+) , Ba(2+) , and Cd(2+) to Ca-free horseradish peroxidase (HRP) enzyme was investigated using UV/VIS spectrophotometry and molecular-mechanic (MM) calculations. According to the obtained binding and thermodynamic parameters, trend of the relative binding affinities of these divalent metal cations was found to be: Ca(2+) >Cd(2+) >Mg(2+) >Ba(2+) . Binding analysis based on Scatchard and Hill models showed positive cooperativity effect between the two distal and proximal binding sites. Furthermore, kinetics of binding and reconstitution process was examined (using relaxation-time method) for binding of Ca(2+) (as the typical metal ion) to Ca-free HRP, which was found a second-order type having a two-step mechanism involving fast formation of Ca-free HRP/1?Ca(2+) as the kinetic intermediate in step 1. Finally, by means of MM calculations, the comparative stability energies were evaluated for binding of M(2+) metal cations to Ca-free HRP. Based on MM calculations, preferential binding of Ca(2+) ion was occurred on distal and proximal binding sites of Ca-free HRP associated with higher stability energies (E(total) ). Indeed, among the divalent metal ions, Ca(2+) with the highest binding affinity (maximum value of K(bin) and minimum value of ΔG$\rm{{_{bin}^{0}}}$), maximum value of exothermic binding enthalpy, and stability energies stabilizes the HRP structure along with an optimized catalytic activity.     

Fluorescence study of the interaction of Suwannee River fulvic acid with metal ions and Al3+-metal ion competition

In this study emission and synchronous-scan fluorescence spectroscopy have been used to investigate the interaction of the class A (oxygen seeking 'hard acid') metal Al(3+), with Suwannee River fulvic acid (SRFA), as well as competition between Al(3+) and several other metal ions (Ca(2+), Mg(2+), Cu(2+), Pd(2+), La(3+), Tb(3+) and Fe(3+)) for binding sites on SRFA. Of the four metal ions possessing very similar (and relatively low) ionic indices (Ca(2+), Mg(2+), Cu(2+) and Pd(2+)) only the latter two paramagnetic ions significantly quenched SRFA fluorescence emission intensity. Of the four metal ions possessing very similar (and relatively low) covalent indices (Ca(2+), Mg(2+), La(3+) and Tb(3+)) only the last paramagnetic ion (Tb(3+)) significantly quenched SRFA fluorescence. None of these metals was able to significantly compete with SRFA-bound Al(3+).Fe(3+), which differs substantially from all of the other metals examined in this study in that it possesses a relatively high ionic index (but not as high as Al(3+)) and a relatively low covalent index (but not as low as Al(3+)), was able not only to quench SRFA fluorescence but also to compete (at least to some extent) with SRFA-bound Al(3+). Synchronous-scan fluorescence SRFA spectra taken in the absence and presence of Fe(3+) and/or Al(3+) support the view that these two metal ions can compete for sites on SRFA. The results of these fluorescence experiments further confirm the Al(3+), and metal ions that have electronic properties somewhat similar to Al(3+) (such as Fe(3+)) are somewhat unique in their ability to interact strongly with binding sites on fulvic acids.     

The role of interfacial binding in the activation of Streptomyces chromofuscus phospholipase D by phosphatidic acid

The Streptomyces chromofuscus phospholipase D (PLD) cleavage of phosphatidylcholine in bilayers can be enhanced by the addition of the product phosphatidic acid (PA). Other anionic lipids such as phosphatidylinositol, oleic acid, or phosphatidylmethanol do not activate this PLD. This allosteric activation by PA could involve a conformational change in the enzyme that alters PLD binding to phospholipid surfaces. To test this, the binding of intact PLD and proteolytically cleaved isoforms to styrene divinylbenzene beads coated with a phospholipid monolayer and to unilamellar vesicles was examined. The results indicate that intact PLD has a very high affinity for PA bilayers at pH >/= 7 in the presence of EGTA that is weakened as Ca(2+) or Ba(2+) are added to the system. Proteolytically clipped PLD also binds tightly to PA in the absence of metal ions. However, the isolated catalytic fragment has a considerably weaker affinity for PA surfaces. In contrast to PA surfaces, all PLD forms exhibited very low affinity for PC interfaces with an increased binding when Ba(2+) was added. All PLD forms also bound tightly to other anionic phospholipid surfaces (e.g. phosphatidylserine, phosphatidylinositol, and phosphatidylmethanol). However, this binding was not modulated in the same way by divalent cations. Chemical cross-linking studies suggested that a major effect of PLD binding to PA.Ca(2+) surfaces is aggregation of the enzyme. These results indicate that PLD partitioning to phospholipid surfaces and kinetic activation are two separate events and suggest that the Ca(2+) modulation of PA.PLD binding involves protein aggregation that may be the critical interaction for activation.     

Cation charge and size selectivity of the C2 domain of cytosolic phospholipase A(2).

C2 domains regulate numerous eukaryotic signaling proteins by docking to target membranes upon binding Ca(2+). Effective activation of the C2 domain by intracellular Ca(2+) signals requires high Ca(2+) selectivity to exclude the prevalent physiological metal ions K(+), Na(+), and Mg(2+). The cooperative binding of two Ca(2+) ions to the C2 domain of cytosolic phospholipase A(2) (cPLA(2)-alpha) induces docking to phosphatidylcholine (PC) membranes. The ionic charge and size selectivities of this C2 domain were probed with representative mono-, di-, and trivalent spherical metal cations. Physiological concentrations of monovalent cations and Mg(2+) failed to bind to the domain and to induce docking to PC membranes. Superphysiological concentrations of Mg(2+) did bind but still failed to induce membrane docking. In contrast, Ca(2+), Sr(2+), and Ba(2+) bound to the domain in the low micromolar range, induced electrophoretic mobility shifts in native polyacrylamide gels, stabilized the domain against thermal denaturation, and induced docking to PC membranes. In the absence of membranes, the degree of apparent positive cooperativity in binding of Ca(2+), Sr(2+), and Ba(2+) decreased with increasing cation size, suggesting that the C2 domain binds two Ca(2+) or Sr(2+) ions, but only one Ba(2+) ion. These stoichiometries were correlated with the abilities of the ions to drive membrane docking, such that micromolar concentrations of Ca(2+) and Sr(2+) triggered docking while even millimolar concentrations of Ba(2+) yielded poor docking efficiency. The simplest explanation is that two bound divalent cations are required for stable membrane association. The physiological Ca(2+) ion triggered membrane docking at 20-fold lower concentrations than Sr(2+), due to both the higher Ca(2+) affinity of the free domain and the higher affinity of the Ca(2+)-loaded domain for membranes. Kinetic studies indicated that Ca(2+) ions bound to the free domain are retained at least 5-fold longer than Sr(2+) ions. Moreover, the Ca(2+)-loaded domain remained bound to membranes 2-fold longer than the Sr(2+)-loaded domain. For both Ca(2+) and Sr(2+), the two bound metal ions dissociate from the protein-membrane complex in two kinetically resolvable steps. Finally, representative trivalent lanthanide ions bound to the domain with high affinity and positive cooperativity, and induced docking to PC membranes. Overall, the results demonstrate that both cation charge and size constraints contribute to the high Ca(2+) selectivity of the C2 domain and suggest that formation of a cPLA(2)-alpha C2 domain-membrane complex requires two bound multivalent metal ions. These features are proposed to stem from the unique structural features of the metal ion-binding site in the C2 domain.     

Energetics and mechanism of Ca2+ displacement by lanthanides in a calcium binding protein

The displacement of Ca(2+) by trivalent lanthanide ions (Tm(3+)) in a protozoan (Entamoeba histolytica) Ca(2+) binding protein has been studied by NMR and isothermal calorimetry (ITC). The study provides a basis for understanding the behavior of lanthanides when used as a substitute for Ca(2+), the pattern of sequential binding, the structural changes involved, the range and magnitude of paramagnetic interaction, and the associated energetics and mechanism. The progressive Ca(2+) displacement from site III first, followed by displacement from site II, I, and IV, as observed during the NMR titration experiments, is interpreted in the light of ITC data to provide a deeper insight into the intradomain and, for the first time, interdomain cooperativity and information about the statistical phenomenon involved in it. A theoretical model governing Ca(2+) displacement is provided. The small structural changes involved in Ca(2+) displacement by a diamagnetic lanthanide (La(3+)) has also been monitored.     

Nonglutamate pore residues in ion selection and conduction in voltage-gated Ca(2+) channels

High-affinity, intrapore binding of Ca(2+) over competing ions is the essential feature in the ion selectivity mechanism of voltage-gated Ca(2+) channels. At the same time, several million Ca(2+) ions can travel each second through the pore of a single open Ca(2+) channel. How such high Ca(2+) flux is achieved in the face of tight Ca(2+) binding is a current area of inquiry, particularly from a structural point of view. The ion selectivity locus comprises four glutamate residues within the channel's pore. These glutamates make unequal contributions to Ca(2+) binding, underscoring a role for neighboring residues in pore function. By comparing two Ca(2+) channels (the L-type alpha(1C), and the non-L-type alpha(1A)) that differ in their pore properties but only differ at a single amino acid position near the selectivity locus, we have identified the amino-terminal neighbor of the glutamate residue in motif III as a determinant of pore function. This position is more important in the function of alpha(1C) channels than in alpha(1A) channels. For a systematic series of mutations at this pore position in alpha(1C), both unitary Ba(2+) conductance and Cd(2+) block of Ba(2+) current varied with residue volume. Pore mutations designed to make alpha(1C) more like alpha(1A) and vice versa revealed that relative selectivity for Ba(2+) over K(+) depended almost solely on pore sequence and not channel type. Analysis of thermodynamic mutant cycles indicates that the motif III neighbor normally interacts in a cooperative fashion with the locus, molding the functional behavior of the pore.     

Artificial lipid-protein complexes accelerate cholesterol crystallisation in model bile

Cholesterol gallstone disease is one of the major health problems in the world. Substances which can affect the crystallisation of cholesterol from human bile have been given considerable attention. Various substances (among them natural lipid-protein complexes) have been tested for cholesterol crystallisation-promoting activity. Various artificial lipid-albumin complexes have been prepared of which taurodeoxycholate-human serum albumin-calcium ions (TDC-HSA-Ca(2+)) had the highest cholesterol crystallisation-promoting activity. This cholesterol crystallisation-promoting activity is similar to that for the lipid-protein complex isolated from native human bile [concanavalin A nonbinding fraction (con A(-) fraction)]. Addition of cholesterol to the TDC-HSA-Ca(2+) complex further increased the cholesterol crystallisation-promoting activity whereas the addition of lecithin had an opposite effect. The interaction of individual components of the TDC-HSA-Ca(2+) complex was followed using several methods. A new effect of Ca(2+) ions (increase in the number of binding sites for bile salts) on the interaction of TDC with HSA was found by equilibrium dialysis. Interaction of TDC with albumin and Ca(2+) did not induce any modification of the secondary structure of albumin. The results of fluorescence spectroscopy may indicate that TDC is at least partially bound to not essentially fatty acid free HSA somehow via admixtures, probably fatty acids. Difference absorption spectrum of the TDC-HSA-Ca(2+)-cholesterol complex was very similar to that of the "natural" lipid-protein complex (con A(-) fraction). From the three drugs with different albumin binding characteristics, only sulphadimethoxin had an observable effect on the cholesterol crystallisation-promoting activity. The action of the TDC-HSA-Ca(2+) complex decreased significantly after the addition of sulphadimethoxin. The addition of TDC modified the absorption spectrum of the sulphadimethoxin-HSA-Ca(2+) complex. It can be suggested that the complex of HSA with bile salts (TDC mainly) and Ca(2+) forms a nucleation centre for cholesterol crystallisation in bile.     

Study of the influence of metal ions on tRNA(Phe) thermal unfolding equilibria by UV spectroscopy and multivariate curve resolution

The influence of metal ions (Na(+), Mg(2+) and Cd(2+)) on the thermal unfolding of phenylalanine transfer ribonucleic acid (tRNA(Phe)) was studied by UV spectroscopy-monitored melting experiments. Absorbance data were obtained during the unfolding process in the range 220-340 nm and later analyzed by a multivariate curve resolution approach (MCR-ALS) based on factor analysis. This procedure determines the number of spectroscopically distinct conformations present during the unfolding process and reveals their concentration profiles and pure spectra, without any initial assumption having to be made about the number of steps in the unfolding pathway. From the concentration profiles and pure spectra, information such as T(m) values can be recovered. The results were compared with those obtained previously in spectroscopic and calorimetric unfolding experiments, showing that the multivariate approach recovers information that complements that obtained in traditional spectroscopic melting experiments.