Binding of alkaline cations to the double-helical form of gramicidin.

Gramicidin is a polypeptide antibiotic that forms monovalent cation-specific channels in membrane environments. In organic solvents and in lipids containing unsaturated fatty acid chains, it forms a double-helical "pore" structure, in which two monomers are intertwined. This form of gramicidin can bind two cations inside its lumen, and the crystal structures of both an ion complex and an ion-free form have been determined. In this study, we have used circular dichroism (CD) spectroscopy to examine the binding mechanism and the binding constants (K1 and K2) of cations to gramicidin in the double helical form in methanol solution. The dramatic change in optical rotation in the far-ultraviolet CD spectrum of gramicidin provides a useful tool for monitoring the binding. The binding mechanism appears to involve a large conformation change associated with the binding of ions to the first of the two sites. The calculated values for the K1 binding constants for alkaline cations are considerably smaller than the K2 binding constants. The order of binding affinity for alkaline cations is similar to that for the helical dimer "channel" form of gramicidin, i.e., Cs+ approximately Rb+ > > K+ > Li+, but in comparison to the helical dimer form, the binding to double-helical dimers is dominated by a cation size-dependent conformational change in the gramicidin structure.     

Competitive Calcium Binding: Implications for Dendritic Calcium Signaling

Action potentials evoke calcium transients in dendrites of neocortical pyramidal neurons with time constants of <100 ms at physiological temperature. This time period may not be sufficient for inflowing calcium ions to equilibrate with all present Ca²⁺-binding molecules. We therefore explored nonequilibrium dynamics of Ca²⁺ binding to numerous Ca²⁺ reaction partners within a dendritelike compartment using numerical simulations. After a brief Ca²⁺ influx, the reaction partner with the fastest Ca²⁺ binding kinetics initially binds more Ca²⁺ than predicted from chemical equilibrium, while companion reaction partners bind less. This difference is consolidated and may result in bypassing of slow reaction partners if a Ca²⁺ clearance mechanism is active. On the other hand, slower reaction partners effectively bind Ca²⁺ during repetitive calcium current pulses or during slower Ca²⁺ influx. Nonequilibrium Ca²⁺ distribution can further be enhanced through strategic placement of the reaction partners within the compartment. Using the Ca²⁺ buffer EGTA as a competitor of fluo-3, we demonstrate competitive Ca²⁺ binding within dendrites experimentally. Nonequilibrium calcium dynamics is proposed as a potential mechanism for differential and conditional activation of intradendritic targets.     

The solution conformations of gramicidin A and its analogs

The conformational states in dioxane and ethanol of gramicidin A and of analogs varying in chain length and amino acid sequence have been studied. Infrared, CD, and polarization of fluorescence spectra of the peptides were measured, from which dimerization constants were determined and spectral characteristics of the monomeric and dimeric states obtained. Resonance splitting of the amide I ir band has been calculated for all gramicidin A models proposed earlier. Detailed comparison of the experimental and computed spectra showed that the four dimeric gramicidin species present in solution are predominantly antiparallel double ?ππ_ld helices in equilibrium with smaller amounts of head-to-head associated π_LD helices. The gramicidin A monomer was found to be a π_LD^4.4 helix in dioxane. For each conformational form the number of residues per turn and the helical sense were determined. The relationship between the amino acid sequence and the structure and stability of the dimer in the series of gramicidin A and its analogs is discussed. The above findings are rationalized in terms of the membrane channel properties of gramicidin A, in particular the conformational rearrangements occurring during the passage of metal ions through the channel and also the differences in conformation of the antibiotic in nonpolar solutions and in the membrane.     

Point amino acid substitutions in the Ca2+-binding sites of recoverin: II. The unusual behavior of the protein upon the binding of calcium ions

The structural properties of myristoylated forms of recombinant recoverin of the wild type and of its mutants with damaged second and/or third Ca²⁺-binding sites were studied by fluorimetry and circular dichroism. The interaction of wild-type recoverin with calcium ions was shown to induce unusual structural rearrangements in its molecule. In particular, protein binding with Ca²⁺ ions results in an increase in the mobility of the environment of Trp residues, in hydrophobicity, and in thermal stability (its thermal transition shifts by 15°C to higher temperatures) but has almost no effect on its secondary structure. Similar structural changes induced by Ca²⁺ are also characteristic of the -EF2 mutant of recoverin whose second Ca²⁺-binding site is modified and cannot bind calcium ions. The structural properties of the -EF3 and -EF2,3 mutants (whose third or simultaneously second and third Ca²⁺-binding sites, respectively, are modified and damaged) are practically indifferent to the presence of calcium ions. For the communication I, see [1].     

Studies on the interaction of cholesterol with soluble and insoluble elastins

The exchange of ³H-cholesterol in an aqueous solution of taurodeoxycholate with insoluble elastin and kappa-elastin peptides has been quantitatively studied. At higher cholesterol concentrations, and in similar experimental conditions, 0.3 μg and 0.2 μg of cholesterol could be adsorbed, respectively, on to 1 mg insoluble elastin and 1 mg kappa-elastin coacervate. Therefore, the above amounts of cholesterol fixed per mg elastin represent minimal estimates. The replacement of Na⁺ ions by Ca²⁺ ions increased the cholesterol binding both of fibrous and of soluble elastins. The binding curves tend towards a limiting value in the presence of Na⁺, but no saturation effect was observed in the presence of Ca²⁺. The replacement of sodium ions by calcium ions in the taurodeoxycholate solutions also lowered the coacervation temperature of the kappa-elastin peptides. Fibrous elastin retained more cholesterol at 65°C than at 37°C, suggesting the importance of hydrophobic interactions in cholesterol elastin association. These results suggest that conformational changes could be induced in both soluble and insoluble forms of elastin by calcium ions increasing their affinity for cholesterol. This enhancement of cholesterol fixation by elastin in the presence of Ca²⁺ ions may well have a physiopathological importance. So does also the fact that elastin-peptides exhibit a higher affinity in the presence of Ca²⁺ for cholesterol than insoluble elastin, suggesting the possibility of increased cholsterol (and calcium) fixation as elastin is degraded in situ by elastases.     

The effect of calcium (II) on the binding of anticoagulation factor I with activated coagulation factor X

Anticoagulation factor I (ACF I) from the venom of Agkistrodon acutus forms a 1:1 complex with activated coagulation factor X (FXa) in a Ca²⁺-dependent fashion and thereby prolongs the clotting time. In the present study, the dependence of the binding of ACF I with FXa on the concentration of Ca²⁺ ions was quantitatively analyzed by HPLC, and the result showed that the maximal binding of ACF I to FXa occurred at concentration of Ca²⁺ ions of about 1 mM. The binding of Ca²⁺ ions to ACF I was investigated by equilibrium dialysis and two Ca²⁺-binding sites with different affinities were identified. At pH 7.6, the apparent association constants K₁ and K₂ for these two sites were (1.8 ± 0.5) × 10⁵ and (2.7 ± 0.6) × 10⁴ M^–1 (mean ± SE, n = 4), respectively. It was evident from the observation of Ca²⁺-induced changes in the intrinsic fluorescence of ACF I that ACF I underwent a conformational change upon binding of Ca²⁺ ions. The occupation of both Ca²⁺-binding sites in ACF I required a concentration of Ca²⁺ ions of about 1 mM, which is equal to the effective concentration of Ca²⁺ ions required both for maximal binding of ACF I to FXa and for the maximal enhancement of emission fluorescence of ACF I. It could be deduced from these results that the occupation of both Ca²⁺-binding sites in ACF I with Ca²⁺ ions and subsequent conformational rearrangement might be essential for the binding of ACF I to FXa.     

Transformation of (Ca + Mg)ATPase of calmodulin-depleted erythrocyte membranes into a high Ca-affinity form by Ca

Specific activity and Ca²⁺-affinity of (Ca²⁺+Mg²⁺)ATPase of calmodulin-depleted ghosts progressively increase during preincubation with 0.1–2 mM Ca²⁺. Concomitantly, the increment in ATPase activity caused by calmodulin and the binding of calmodulin to ghosts decrease. The effects of calcium ions are abolished by the addition of calmodulin. ATP protects the enzyme from a Ca²⁺-dependent decrease of the maximum activity but does not seem to influence the Ca²⁺-dependent transformation of the low Ca²⁺-affinity enzyme into a high Ca²⁺-affinity form.     

Transformation of (Ca2+ + Mg2+)ATPase of calmodulin-depleted erythrocyte membranes into a high Ca2+-affinity form by Ca2+

Specific activity and Ca²⁺-affinity of (Ca²⁺+Mg²⁺)ATPase of calmodulin-depleted ghosts progressively increase during preincubation with 0.1–2 mM Ca²⁺. Concomitantly, the increment in ATPase activity caused by calmodulin and the binding of calmodulin to ghosts decrease. The effects of calcium ions are abolished by the addition of calmodulin. ATP protects the enzyme from a Ca²⁺-dependent decrease of the maximum activity but does not seem to influence the Ca²⁺-dependent transformation of the low Ca²⁺-affinity enzyme into a high Ca²⁺-affinity form.     

Ca2+ binding and translocation by the sarcoplasmic reticulum ATPase: Functional and structural considerations

Three experimental systems are described including sarcoplasmic reticulum (SR) vesicles, reconstituted proteoliposomes, and recombinant protein obtained by gene transfer and expression in foreign cells. It is shown that the Ca²⁺ ATPase of sarcoplasmic reticulum (SR) includes an extramembranous globular head which is connected through a stalk to a membrane bound region. Cooperative binding of two calcium ions occurs sequentially, within a channel formed by four clustered helices within the membrane bound region. Destabilization of the helical cluster is produced following enzyme phosphorylation by ATP at the catalytic site in the extramembranous region. The affinity and orientation of the Ca²⁺ binding site are thereby changed, permitting vectorial dissociation of bound Ca²⁺ against a concentration gradient. A long range linkage between phosphorylation and Ca²⁺ binding sites is provided by an intervening peptide segment that retains high homology in cation transport ATPases, and whose function is highly sensitive to mutational perturbations.     

The modification of the unidirectional calcium fluxes of sarcoplasmic reticulum vesicles by monovalent cation ionophores

Calcium uptake by rabbit skeletal muscle sarcoplasmic reticulum vesicles in phosphate-containing media exhibits time-dependent changes that arise from changing rates of calcium influx and efflux. The monovalent cation ionophore gramicidin, added before the start of the calcium uptake reaction, delayed the spontaneous calcium release that normally occurred after approx. 6 min in such reactions; the rate of calcium efflux was inhibited while calcium influx was little affected. Under these conditions, Ca²⁺-activated ATPase activity could remain unaltered.Gramicidin stimulated calcium uptake irrespective of the presence of a K⁺ gradient across the vesicle membrane. Valinomycin stimulated calcium uptake in a manner similar to that for gramicidin even in an NaCl-containing medium lacking potassium. Thus, dissipation of a transmembrane K⁺ gradient is unlikely to account for the effects of these ionophores on the spontaneous changes in calcium flux rates.Addition of gramicidin to partially calcium-filled vesicles inhibited the phase of spontaneous calcium reuptake because both calcium influx and efflux were inhibited. Addition of gramicidin to partially calcium-filled vesicles in the presence of a water-soluble protein, such as bovine serum albumin, creatine kinase or pyruvate kinase, markedly stimulated calcium uptake. This stimulatory effect was due primarily to inhibition of calcium efflux, calcium influx being minimally influenced by the ionophore.After cleavage of the 100 000 dalton ATPase to 50 000 dalton fragments, which was not associated with changes in Ca²⁺-activated ATPase activity or initial calcium uptake rate, gramicidin increased rather than decreased calcium content when added to vesicles after the initial maximum in calcium content. Thus, the ability of monovalent cation ionophores to block calcium efflux from calcium-filled vesicles may reflect their interaction with a portion of the Ca²⁺-activated ATPase protein.     

Tertiary structure and energy coupling in Ca2+-pump system

Europium luminescence from europium bound to sarcoplasmic reticulum (Ca²⁺ Mg²⁺)-ATPase indicates that there are two high affinity calcium binding sites. Furthermore, the two calcium ions at the binding sites are highly coordinated by the protein as the number of H₂O molecules surrounding the Ca²⁺ ions are 3 and 0.5. In the presence of ATP, calcium ions are occluded even further down to 2 and zero H2O molecules, respectively. The Ca²⁺ - Ca²⁺ intersite distance is estimated to be 8–9 Å and the average distance from the Ca²⁺ sites to CrATP is about 18 Å.Digestion of the (Ca²⁺ + Mg²⁺)-ATPase at the T₂ site (Arg 198) causes uncoupling of Ca²⁺-transport from ATPase activity while calcium occlusion due to E₁-P formation remains unchanged. Further tryptic digestion beyond T₂ and in the presence of ATP diminishes Ca²⁺ occlusion to zero while 50% of the ATPase hydrolytic activity remains. Tryptic digestion beyond T₂ and in the absence of ATP diminishes ATPase hydrolytic activity to 50% of normal while Ca²⁺ occlusion remains intact. These data are consistent with a mechanism in which the functional enzyme must be in the dimeric form for occlusion and calcium uptake to occur, but each monomer can hydrolyze ATP.     

Protein solution structure calculations in solution: Solvated molecular dynamics refinement of calbindin D9k

The three-dimensional solution structures of proteins determinedwith NMR-derived constraints are almost always calculated in vacuo. Thesolution structure of (Ca²⁺)_{_2}-calbindinD_9k has been redetermined by new restrained molecular dynamics(MD) calculations that include Ca²⁺ ions and explicit solventmolecules. Four parallel sets of MD refinements were run to provide accuratecomparisons of structures produced in vacuo, in vacuo withCa²⁺ ions, and with two different protocols in a solvent bathwith Ca²⁺ ions. The structural ensembles were analyzed interms of structural definition, molecular energies, packing density,solvent-accessible surface, hydrogen bonds, and the coordination of calciumions in the two binding loops. Refinement including Ca²⁺ ionsand explicit solvent results in significant improvements in the precisionand accuracy of the structure, particularly in the binding loops. Theseresults are consistent with results previously obtained in free MDsimulations of proteins in solution and show that the rMD refinedNMR-derived solution structures of proteins, especially metalloproteins, canbe significantly improved by these strategies.     

The structure of horseradish peroxidase C characterized as a molten globule state after Ca2+ depletion

The structure and activity of native horseradish peroxidase C (HRP) is stabilized by two bound Ca²⁺ ions. Earlier studies suggested a critical role of one of the bound Ca²⁺ ions but with conflicting conclusions concerning their respective importance. In this work we compare the native and totally Ca²⁺-depleted forms of the enzyme using pH-, pressure-, viscosity- and temperature-dependent UV absorption, CD, H/D exchange-FTIR spectroscopy and by binding the substrate benzohydroxamic acid (BHA). We report that Ca²⁺-depletion does not change the alpha helical content of the protein, but strongly modifies the tertiary structure and dynamics to yield a homogeneously loosened molten globule-like structure. We relate observed tertiary changes in the heme pocket to changes in the dipole orientation and coordination of a distal water molecule. Deprotonation of distal His42, linked to Asp43, itself coordinated to the distal Ca²⁺, perturbs a H-bonding network connecting this Ca²⁺ to the heme crevice that involves the distal water. The measured effects of Ca²⁺ depletion can be interpreted as supporting a structural role for the distal Ca²⁺ and for its enhanced significance in finetuning the protein structure to optimize enzyme activity.     

Ruthenium red as a resonance Raman probe of Ca2+ binding sites in biological materials.

We have observed substantial changes in the resonance Raman spectrum of ruthenium red when it is added to calcium ion binding molecules and organelles, including proteins, phospholipids, chelating agents, and intact mitochondria. The addition of Ca²⁺ ions can reverse these observed spectral changes. In the case of cytochrome c, ruthenium red binding varies with oxidation state in a manner parallel to that for Ca²⁺ binding. The resonance Raman spectrum of a ruthenium red-phospholipid complex shows differences from that of a ruthenium red-protein complex, enabling us to distinguish between binding to these different classes of molecules. Our studies suggest that the primary constituent of the low-affinity Ca²⁺ binding sites in mitochondria is cardiolipin.     

Effects of Ca2+ and calmodulin on adenylyl cyclase activity in sheep olfactory epithelium

Sheep olfactory epithelium contains an adenylyl cyclase which is stimulated by many but not all odorants. Here we report that this enzyme is activated by calmodulin in a dose-dependent manner, and that calcium ions are required for this response. Odorant stimulation of adenylyl cyclase is unaffected by the complex Ca²⁺/calmodulin, as suggested by the results obtained both in Ca²⁺/calmodulin-depleted membranes and under calmodulin antagonist treatment; this confirms the prediction that the Ca²⁺ binding protein and odorants stimulate the olfactory adenylyl cyclase through parallel mechanisms. The persistent activation of the regulatory component of adenylyl cyclase by GppNHp does not alter the response of the enzyme to either odorant or Ca²⁺/calmodulin. In sheep olfactory epithelium a cAMP-phosphodiesterase activity is also present, which is highly inhibited by IBMX and aminophylline, searcely by RO 20-1724, and unaffected by Ca²⁺/calmodulin. The modulatory role exerted by calcium on cAMP system in sheep olfactory signal transduction is discussed.     

Sorption of alkaline earth metal ions Ca2+ and Mg2+ on lyocell fibres

Ca²⁺ and Mg²⁺ content of cellulose fibres is of relevance for a wide range of applications e.g. textile processing, pulp/paper, food. Sorption of Ca²⁺ and Mg²⁺ ions were found on lyocell type regenerated cellulose fibres. Higher affinity was found for Ca²⁺ ions compared to Mg²⁺ ions. At pH 9, fibre saturation was observed at a calcium binding capacity of 18–20 mmol/kg. A carboxylic group content of 18 mmol COOH per kg fibre material was determined based on the Methylene Blue absorption. This indicates a 1:1 molar stoichiometry between the carboxylic groups present in the fibres and the bound Ca²⁺ ions. Thus it is proposed that the salt in fibre shows the general composition (Cell-O^? Ca²⁺ X^?), X^? being an anion bound in the salt to achieve charge neutrality.The sorption of Ca²⁺ also can be demonstrated by complex formation with 1,2-dihydroxy-9,10-anthraquinone (alizarin) which forms a red-violet Ca²⁺-complex. Colour fixation thus can be used as an indicator for the Ca²⁺-ions bound in the fibre.     

Interaction of Calcium Ion with Bovine Caseins

In order to clarify the interaction of calcium ion with casein, the volume change associated with the interaction was measured by dilatometric procedures. When CaCl₂ was added to the casein solutions at neutral pH, a volume increase occurred and reached a constant saturated value of about 700 ml per 10⁶ g protein with increasing CaCl₂ concentrations for whole-, α_s- and β-casein solutions, but there was no volume change for κ-casein solution. On the other hand, the binding of calcium ion to the casein fractions was determined by a gel filtration procedure at pH 6.0 to 9.0. The number of Ca²⁺ ions bound to the caseins increased with the CaCl₂ concentration and pH value, and the relative order of binding capacities for the caseins was: α_s-casein > whole-casein > β-casein > κ-casein.

It was found that the volume changes obtained by the dilatometry were smaller than the calculated volume increases based on the assumption that these are caused by the binding of Ca²⁺ ion to the caseins. Therefore it is necessary to introduce another factor which reduces the volume increase due to the Ca²⁺ ion binding in order to reasonably explain the measured volume changes. At present it is presumed that there occurs the unfolding of peptide chain of casein molecule on Ca²⁺ ion binding, which has been known to decrease the volume of the protein solution.     

Oligomerization of the Polycystin-2 C-terminal Tail and Effects on Its Ca2+-binding Properties

Polycystin-2 (PC2) belongs to the transient receptor potential (TRP) family and forms a Ca²⁺-regulated channel. The C-terminal cytoplasmic tail of human PC2 (HPC2 Cterm) is important for PC2 channel assembly and regulation. In this study, we characterized the oligomeric states and Ca²⁺-binding profiles in the C-terminal tail using biophysical approaches. Specifically, we determined that HPC2 Cterm forms a trimer in solution with and without Ca²⁺ bound, although TRP channels are believed to be tetramers. We found that there is only one Ca²⁺-binding site in the HPC2 Cterm, located within its EF-hand domain. However, the Ca²⁺ binding affinity of the HPC2 Cterm trimer is greatly enhanced relative to the intrinsic binding affinity of the isolated EF-hand domain. We also employed the sea urchin PC2 (SUPC2) as a model for biophysical and structural characterization. The sea urchin C-terminal construct (SUPC2 Ccore) also forms trimers in solution, independent of Ca²⁺ binding. In contrast to the human PC2, the SUPC2 Ccore contains two cooperative Ca²⁺-binding sites within its EF-hand domain. Consequently, trimerization does not further improve the affinity of Ca²⁺ binding in the SUPC2 Ccore relative to the isolated EF-hand domain. Using NMR, we localized the Ca²⁺-binding sites in the SUPC2 Ccore and characterized the conformational changes in its EF-hand domain due to trimer formation. Our study provides a structural basis for understanding the Ca²⁺-dependent regulation of the PC2 channel by its cytosolic C-terminal domain. The improved methodology also serves as a good strategy to characterize other Ca²⁺-binding proteins.     

Binding of calcium is sensed structurally and dynamically throughout the second calcium‐binding domain of the sodium/calcium exchanger

We report the effects of Ca²⁺ binding on the backbone relaxation rates and chemical shifts of the AD and BD splice variants of the second Ca²⁺‐binding domain (CBD2) of the sodium–calcium exchanger. Analysis of the Ca²⁺‐induced chemical shifts perturbations yields similar K_D values of 16–24 μM for the two CBD2‐AD Ca²⁺‐binding sites, and significant effects are observed up to 20 Å away. To quantify the Ca²⁺‐induced chemical shift changes, we performed a comparative analysis of eight Ca²⁺‐binding proteins that revealed large differences between different protein folds. The CBD2 ¹⁵N relaxation data show the CBD2‐AD Ca²⁺ coordinating loops to be more rigid in the Ca²⁺‐bound state as well as to affect the FG‐loop located at the opposite site of the domain. The equivalent loops of the CBD2‐BD splice variant do not bind Ca²⁺ and are much more dynamic relative to both the Ca²⁺‐bound and apo forms of CBD2‐AD. A more structured FG‐loop in CBD2‐BD is suggested by increased S² order parameter values relative to both forms of CBD2‐AD. The chemical shift and relaxation data together indicate that, in spite of the small structural changes, the Ca²⁺‐binding event is felt throughout the molecule. The data suggest that the FG‐loop plays an important role in connecting the Ca²⁺‐binding event with the other cytosolic domains of the NCX, in line with in vivo and in vitro biochemical data as well as modeling results that connect the CBD2 FG‐loop with the first Ca²⁺‐binding domain of NCX. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Binding of thallium and other cations to the gramicidin A channel. Equilibrium dialysis study of gramicidin in phosphatidylcholine vesicles

Gramicidin A is a linear peptide antibiotic which forms dimer transmembrane channels selective for small monovalent cations, including thallium ions (Tl⁺) which are strongly bound. While there is great interest in the number of ion-binding sites per channel and the affinities of the sites for the various cations, measurements of the kinetics of ion permeation yield these equilibrium parameters only as indirect estimates dependent on the model assumed for the channel. Sonicated lipid vesicles. containing 1 mole of gramicidin per 30 moles of dimyristoylphosphatidylcholine. can be prepared with 5 mm-gramicidin. Evidence from our previous spectroscopic studies strongly supports the belief that this gramicidin is in the form of symmetrical dimer channels. Lipid vesicles containing gramicidin were dialyzed against control vesicles without gramicidin in the presence of a constant amount of radioactive ²⁰¹Tl⁺ and increasing amounts of non-radioactive Tl⁺. The ratio of ²⁰¹Tl⁺ free in solution to ²⁰¹Tl⁺ bound to the channel was measured after equilibrium (≥ 48 h) at 23 °C, and this ratio was plotted as a function of the free Tl⁺ concentration. The inverse of the slope yielded 0.8 to 1.1 for the maximum number of simultaneously occupied highest affinity sites per channel, and the inverse of the intercept yielded a highest affinity constant of 500 to 1000 m^?1 for each site. It appears that direct electrostatic repulsion prevents ions from binding simultaneously to the identical channel ends for thallium ion concentrations up to 20 mm. Estimates of the highest affinity constants for Rb⁺ and Na⁺ were also obtained.