共查询到20条相似文献,搜索用时 15 毫秒
1.
Xiaolong Xu Liyun Zhang Dengke Shen Hao Wu Lili Peng Jiehua Li 《Journal of biological inorganic chemistry》2009,14(4):559-571
Anticoagulation factor I (ACF I) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X (FXa)-binding protein that binds in a Ca2+-dependent fashion with marked anticoagulant activity. The thermodynamics of the binding of alkaline earth metal ions to ACF
I and the effects of alkaline earth metal ions on the guanidine hydrochloride (GdnHCl)-induced unfolding of ACF I and the
binding of ACF I to FXa were studied by isothermal titration calorimetry, fluorescence, circular dichroism, and surface plasmon
resonance, respectively. The results indicate that the ionic radii of the cations occupying Ca2+-binding sites in ACF I crucially affect the binding affinity of ACF I for alkaline earth metal ions as well as the structural
stability of ACF I against GdnHCl denaturation. Sr2+ and Ba2+, with ionic radii larger than the ionic radius of Ca2+, can bind to Ca2+-free ACF I (apo-ACF I), while Mg2+, with an ionic radius smaller than that of Ca2+, shows significantly low affinity for the binding to apo-ACF I. All bindings of Ca2+, Sr2+, and Ba2+ ions in two sites of ACF I are mainly enthalpy-driven and the entropy is unfavorable for them. Sr2+-stabilized ACF I exhibits slightly lower resistance to GdnHCl denaturation than Ca2+–ACF I, while Ba2+-stabilized ACF I exhibits much lower resistance to GdnHCl denaturation than Ca2+–ACF I. Mg2+ and Sr2+, with ionic radii close to that of Ca2+, can bind to FXa and therefore also induce the binding of ACF I to FXa, whereas Ba2+, with a much larger ionic radius than Ca2+, cannot support the binding of ACF I with FXa. Our observations suggest that bindings of Ca2+, Sr2+, and Ba2+ ions in two sites of ACF I increase the structural stability of ACF I, but these bindings are not essential for the binding
of ACF I with FXa, and that the binding of Mg2+, Ca2+, and Sr2+ ions to FXa may be essential for the recognition between FXa and ACF I. 相似文献
2.
Anticoagulation factor I (ACF I) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein with marked anticoagulant activity. Present studies show that holo-ACF I assumes a compactly folded structure in the range of pH 5–6, in which the most interior Trp residues and quenchers are adjacent. Tb3+ ions can completely replace both Ca2+ ions in holo-ACF I, as determined by equilibrium dialysis. Although the two Tb3+ ions in Tb3+-ACF I have slightly different luminescence efficiencies, both have similar quenching effects on the intrinsic fluorescence, suggesting that probably there are same numbers of Trp residues close to both Tb3+-binding sites. Two Tb3+-binding sites with similar apparent Tb3+ association constant values, (1.69 ± 0.02) × 107 M–1 and (1.42 ± 0.01) × 107 M–1, respectively, were further identified through Tb3+ fluorescence titration. In addition, it has been confirmed from the titration of holo-ACF I and Tb3+-ACF I with NBS that only interior Trp residues are involved in the energy transfer to Tb3+ ions and that all accessible Trp residues located in the surface of holo-ACF I have similar affinity to NBS, while those located in the surface of Tb3+-ACF I have two different kinds of affinity to NBS, which strongly suggests a conformational change of holo-ACF I upon substitution of Tb3+ for Ca2+. The results show that although the Tb3+-altered conformation of ACF I cannot support the binding of Tb3+-ACF I with FXa, determined by nondenaturing PAGE, Tb3+ ions are effective and useful fluorescence probes to analyze the structures and properties of Ca2+-binding sites in ACF I. 相似文献
3.
Dengke Shen Xiaolong Xu Hao Wu Lili Peng Yan Zhang Jiajia Song Qingde Su 《Journal of biological inorganic chemistry》2011,16(4):523-537
Anticoagulation factor II (ACF II) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X (FXa)-binding protein with both anticoagulant and hypotensive activities. The thermodynamics
of the binding of alkaline earth metal ions to ACF II and their effects on the stability of ACF II and the binding of ACF
II to FXa were investigated by isothermal titration calorimetry, fluorescence, differential scanning calorimetry, and surface
plasmon resonance. The binding of ACF II to FXa does not have an absolute requirement for Ca2+. Mg2+, Sr2+, and Ba2+ can induce the binding of ACF II to FXa. The radii of the cations bound in ACF II crucially affect the binding affinity of
ACF II for cations and the structural stability of ACF II against guanidine hydrochloride and thermal denaturation, whereas
the radii of cations bound in FXa markedly affect the binding affinity between ACF II and FXa. The binding affinities of ACF
II for cations and the capacities of metal-induced stabilization of ACF II follow the same trend: Ca2+ > Sr2+ > Ba2+. The metal-induced binding affinities of ACF II for FXa follow the trend Mg2+ > Ca2+ > Sr2+ > Ba2+. Although Mg2+ shows significantly low binding affinity with ACF II, Mg2+ is the most effective to induce the binding of ACF II with FXa. Our observations suggest that in blood the bindings of Ca2+ in two sites of ACF II increase the structural stability of ACF II, but these bindings are not essential for the binding
of ACF II with FXa, and that the binding of Mg2+ and Ca2+ to FXa may be essential for the recognition between FXa and ACF II. Like Ca2+, the abundant Mg2+ in blood also plays an important role in the anticoagulation of ACF II. 相似文献
4.
Pb2+ is known to displace physiologically-relevant metal ions in proteins. To investigate potential relationships between Pb2+/protein complexes and toxicity, data from the protein data bank were analyzed to compare structural properties of Pb2+- and Ca2+-binding sites. Results of this analysis reveal that the majority of Pb2+ sites (77.1%) involve 2-5 binding ligands, compared with 6 ± 2 for non-EF-Hand and 7 ± 1 for EF-Hand Ca2+-binding sites. The mean net negative charge by site (1.7) fell between values noted for non-EF-Hand (1 ± 1) and EF-Hand (3 ± 1). Oxygen is the dominant ligand for both Pb2+ and Ca2+, but Pb2+ binds predominantly with sidechain Glu (38.4%), which is less prevalent in both non-EF-Hand (10.4%) and EF-Hand (26.6%) Ca2+-binding sites. A comparison of binding geometries where Pb2+ has replaced Ca2+ in calmodulin (CaM) and Zn2+ in 5-aminolaevulinic acid dehydratase (ALAD) revealed protein structural changes that appear to be unrelated to ionic displacement. Structural changes observed with CaM may be related to opportunistic binding of Pb2+ in regions of high electrostatic charge, whereas ALAD may bind multiple Pb2+ ions in the active site. These results suggest that Pb2+ adapts to structurally-diverse binding geometries and that opportunistic binding may play an active role in molecular metal toxicity. 相似文献
5.
P. G. Kostyuk S. L. Mironov P. A. Doroshenko V. N. Ponomarev 《The Journal of membrane biology》1982,70(3):171-179
Summary The shifts of current-voltage characteristics of sodium and calcium inward currents produced by changes in the concentration of divalent cations (Mg2+, Ca2+, Sr2+, Ba2+) and in pH of the extracellular solution have been measured on isolated neurons of the molluscHelix pomatia intracellularly perfused with potassium-free solutions. On the basis of these shifts and using Stern's theory (O. Stern, 1924.Z. Electrochem.
30508–516), the binding constants for the ions to charged groups of the outer side of the somatic membrane and the density of the surface charges produced by these groups have been calculated. For groups located in the vicinity of sodium channels we obtainedK
Ca=90±10,K
Sr=60±10,K
Ba=25±5 andK
Mg=16±5m
–1 at pH=7.7 and for groups located in the vicinity of calcium channelsK
Ca=67±10,K
Sr=20±5 andK
Ba=19±5m
–1 at pH=7.0. The same groups bind H+ ions with apparent pK=6.2±0.2 that corresponds toK
H=1.6×106
m
–1. The density of fixed charges near the sodium channels is 0.17±0.05 e/nm2 (pH=7.7) and near the calcium channels is 0.23±0.05 electrons/nm2 (pH=7.0). From the comparison of the obtained values with the data about binding constants of the same ions to different negatively charged phospholipids, a suggestion is made that just the phophatidylserine is responsible for the surface potential of the outer side of the somatic membrane. It was also shown that the presence of this potential results in a change in the concentration of carrier ions near the membrane which affects the maximal values of the corresponding transmembrane currents. 相似文献
6.
7.
Tony Christopeit 《Analytical biochemistry》2009,391(1):39-22
The interactions between Ca2+ and C-reactive protein (CRP) have been characterized using a surface plasmon resonance (SPR) biosensor. The protein was immobilized on a sensor chip, and increasing concentrations of Ca2+ or phosphocholine were injected. Binding of Ca2+ induced a 10-fold higher signal than expected from the molecular weight of Ca2+. It was interpreted to result from the conformational change that occurs on binding of Ca2+. Two sites with different characteristics were distinguished: a high-affinity site with KD = 0.03 mM and a low-affinity site with KD = 5.45 mM. The pH dependencies of the two Ca2+ interactions were different and enabled the assignment of the different sites in the three-dimensional structure of CRP. There was no evidence for cooperativity in the phosphocholine interaction, which had KD = 5 μM at 10 mM Ca2+. SPR biosensors can clearly detect and quantify the binding of very small molecules or ions to immobilized proteins despite the theoretically very low signals expected on binding, provided that significant conformational changes are involved. Both the interactions and the conformational changes can be characterized. The data have important implications for the understanding of the function of CRP and suggest that Ca2+ is an efficient regulator under physiological conditions. 相似文献
8.
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. 相似文献
9.
Christine PeineltHans-Jürgen Apell 《Biophysical journal》2002,82(1):170-181
Electrochromic styryl dyes were used to investigate mutually antagonistic effects of Ca2+ and H+ on binding of the other ion in the E1 and P-E2 states of the SR Ca-ATPase. On the cytoplasmic side of the protein in the absence of Mg2+ a strictly competitive binding sequence, H2E1?HE1?E1?CaE1?Ca2E1, was found with two Ca2+ ions bound cooperatively. The apparent equilibrium dissociation constants were in the order of K1/2(2 Ca) = 34 nM, K1/2(H) = 1 nM and K1/2(H2) = 1.32 μM. Up to 2 Mg2+ ions were also able to enter the binding sites electrogenically and to compete with the transported substrate ions (K1/2(Mg) = 165 μM, K1/2(Mg2) = 7.4 mM). In the P-E2 state, with binding sites facing the lumen of the sarcoplasmatic reticulum, the measured concentration dependence of Ca2+ and H+ binding could be described satisfactorily only with a branched reaction scheme in which a mixed state, P-E2CaH, exists. From numerical simulations, equilibrium dissociation constants could be determined for Ca2+ (0.4 mM and 25 mM) and H+ (2 μM and 10 μM). These simulations reproduced all observed antagonistic concentration dependences. The comparison of the dielectric ion binding in the E1 and P-E2 conformations indicates that the transition between both conformations is accompanied by a shift of their (dielectric) position. 相似文献
10.
O. Gateau-Roesch E. Pavlov A. V. Lazareva E. A. Limarenko C. Levrat N-E. L. Saris P. Louisot G. D. Mironova 《Journal of bioenergetics and biomembranes》2000,32(1):105-110
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 Kd of 8 × 10–6 M, while at pH7.5 this Kd was decreased to 9 × 10–5 M. Bmax 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. 相似文献
11.
Population Shift Underlies Ca2+-induced Regulatory Transitions in the Sodium-Calcium Exchanger (NCX)
Moshe Giladi Reuben Hiller Joel A. Hirsch Daniel Khananshvili 《The Journal of biological chemistry》2013,288(32):23141-23149
In eukaryotic Na+/Ca2+ exchangers (NCX) the Ca2+ binding CBD1 and CBD2 domains form a two-domain regulatory tandem (CBD12). An allosteric Ca2+ sensor (Ca3–Ca4 sites) is located on CBD1, whereas CBD2 contains a splice-variant segment. Recently, a Ca2+-driven interdomain switch has been described, albeit how it couples Ca2+ binding with signal propagation remains unclear. To resolve the dynamic features of Ca2+-induced conformational transitions we analyze here distinct splice variants and mutants of isolated CBD12 at varying temperatures by using small angle x-ray scattering (SAXS) and equilibrium 45Ca2+ binding assays. The ensemble optimization method SAXS analysis demonstrates that the apo and Mg2+-bound forms of CBD12 are highly flexible, whereas Ca2+ binding to the Ca3–Ca4 sites results in a population shift of conformational landscape to more rigidified states. Population shift occurs even under conditions in which no effect of Ca2+ is observed on the globally derived Dmax (maximal interatomic distance), although under comparable conditions a normal [Ca2+]-dependent allosteric regulation occurs. Low affinity sites (Ca1–Ca2) of CBD1 do not contribute to Ca2+-induced population shift, but the occupancy of these sites by 1 mm Mg2+ shifts the Ca2+ affinity (Kd) at the neighboring Ca3–Ca4 sites from ∼ 50 nm to ∼ 200 nm and thus, keeps the primary Ca2+ sensor (Ca3–Ca4 sites) within a physiological range. Thus, Ca2+ binding to the Ca3–Ca4 sites results in a population shift, where more constraint conformational states become highly populated at dynamic equilibrium in the absence of global conformational transitions in CBD alignment. 相似文献
12.
In this study, passive Ca2+ binding was determined in ventricular homogenates (VH) from neonatal (4–6 days) and adult rats, as well as in digitonin-permeabilized adult ventricular myocytes. Ca2+ binding sites, both endogenous and exogenous (Indo-1 and BAPTA) were titrated. Sarcoplasmic reticulum and mitochondrial Ca2+ uptake were blocked by thapsigargin and Ru360, respectively. Free [Ca2+] ([Ca2+]F was measured with Indo-1 and bound Ca2+ ([Ca2+]B) was the difference between [Ca2+]F and total Ca2+. Apparent Ca2+ dissociation constants (Kd) for BAPTA and Indo-1 were increased by 10–20 mg VH protein/ml (from 0.35 to 0.92 μM for Indo-1 and from 0.20 to 0.76 μM for BAPTA) and also by ruthenium red in the case of Indo-1. Titration with successive CaCl2 additions (2.5–10 nmoles) yielded δ[Ca2+]B/δ[Ca2+]F for the sum of [Ca2+]B at all three classes of binding sites. From this function, the apparent number of endogenous sites (Ben) and their Kd (Ken) were determined. Similar Ken values were obtained in neonatal and adult VH, as well as in adult myocytes (0.68 ± 0.14 μM, 0.69 ± 0.13 μM and 0.53 ± 0.10 μM, respectively). However, Ben was significantly higher in adult myocytes than in adult VH (1.73 ± 0.35 versus 0.70 ± 0.12 nmol/mg protein, P < 0.01), which correspond to ∼300 and 213 μmol/l cytosol. This indicates that binding sites are more concentrated in myocytes than in other ventricular components and that Ben determined in VH underestimates cellular Ben by 29%. Although Ben values in nmol/mg protein were similar in adult and neonatal VH (0.69 ± 0.12), protein content was much higher in adult ventricle (125 ± 7 versus 80 ± 1 mg protein/g wet weight, P < 0.01). Expressing Ben per unit cell volume (accounting for fractional mitochondrial volume, and 29% dilution in homogenate), the passive Ca2+ binding capacity at high-affinity sites is ∼300 and 176 mmol/I cytosol in adult and neonatal rat ventricular myocytes, respectively. Additional estimates suggest that passive Ca2+ buffering capacity in rat ventricle increases markedly during the first two weeks of life and that adult levels are attained by the end of the first month. 相似文献
13.
The effect of metal ions on human activated Factor X (Factor Xa) hydrolysis of the chromogenic substrate benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222) was studied utilizing initial rate enzyme kinetics. The divalent metal ions Ca2+, Mn2+, and Mg2+ enhanced Factor Xa amidolytic activity with Km values of 30 μm, 20 μm, and 1.4 mm, respectively. Na+ activation of Factor Xa amidolytic activity was also found. The Km for Na+ activation was 0.31 m. Both the divalent metal ions and Na+ increased the affinity of Factor Xa for S2222 and had no effect on the maximal velocity of the reaction. Other monovalent cations were unable to activate Factor Xa. However, K+ was a competitive inhibitor of the Na+ activation (Ki = 0.14 m). Lanthanide ions inhibited Factor Xa amidolytic activity. Gd3+ inhibition of Factor Xa hydrolysis of S2222 was noncompetitive and had a Ki of 3 μm. The lanthanide ion inhibition could not be reversed by Ca2+ even when Ca2+ was present in a 1000-fold excess over its Km indicating nonidentity of the Factor Xa lanthanide and Ca2+ binding sites. It is concluded that the Factor Xa Ca2+ binding sites have characteristics different from those previously described for the Factor X molecule and that Mg2+, Na+, and K+ may be physiological regulators of Factor Xa activity. 相似文献
14.
The Ca2+-binding properties of placental plasma membranes were studied using a flow dialysis system.Ca2+-binding was not detectable at pH 4.0, but increased at higher pH values to a maximum binding at pH 11.0.Two types of Ca2+-binding sites were identified: high-affinity sites with dissociation constant Ks = 3.1 · 10−5 M and a capacity of 26 nmoles per mg protein; low-affinity sites with Ks = 1.1 · 10−3 M and a capacity of 266 nmoles per mg protein.The affinities of Mg2+ and Sr2+ for the high-affinity sites were 10-fold lower than that of Ca2+, and for the low-affinity sites were 4- and 8-fold lower, respectively.The placental plasma membranes contain sites for Ca2+ with capacity, specificity and affinity within the range reported for other membranes involved in an active transport of Ca2+ (mitochondria, sarcoplasmic reticulum, cardiac microsomes). The presence of high-affinity Ca2+ sites as well as Ca2+-ATPase implicates these membranes in Ca2+ transport from the maternal to the fetal circulation. 相似文献
15.
We have performed microsecond molecular dynamics (MD) simulations to characterize the structural dynamics of cation-bound E1 intermediate states of the calcium pump (sarcoendoplasmic reticulum Ca2+-ATPase, SERCA) in atomic detail, including a lipid bilayer with aqueous solution on both sides. X-ray crystallography with 40 mM Mg2+ in the absence of Ca2+ has shown that SERCA adopts an E1 structure with transmembrane Ca2+-binding sites I and II exposed to the cytosol, stabilized by a single Mg2+ bound to a hybrid binding site I′. This Mg2+-bound E1 intermediate state, designated E1•Mg2+, is proposed to constitute a functional SERCA intermediate that catalyzes the transition from E2 to E1•2Ca2+ by facilitating H+/Ca2+ exchange. To test this hypothesis, we performed two independent MD simulations based on the E1•Mg2+ crystal structure, starting in the presence or absence of initially-bound Mg2+. Both simulations were performed for 1 µs in a solution containing 100 mM K+ and 5 mM Mg2+ in the absence of Ca2+, mimicking muscle cytosol during relaxation. In the presence of initially-bound Mg2+, SERCA site I′ maintained Mg2+ binding during the entire MD trajectory, and the cytosolic headpiece maintained a semi-open structure. In the absence of initially-bound Mg2+, two K+ ions rapidly bound to sites I and I′ and stayed loosely bound during most of the simulation, while the cytosolic headpiece shifted gradually to a more open structure. Thus MD simulations predict that both E1•Mg2+ and E•2K+ intermediate states of SERCA are populated in solution in the absence of Ca2+, with the more open 2K+-bound state being more abundant at physiological ion concentrations. We propose that the E1•2K+ state acts as a functional intermediate that facilitates the E2 to E1•2Ca2+ transition through two mechanisms: by pre-organizing transport sites for Ca2+ binding, and by partially opening the cytosolic headpiece prior to Ca2+ activation of nucleotide binding. 相似文献
16.
Voltage-gated calcium (CaV) channels deliver Ca2+ to trigger cellular functions ranging from cardiac muscle contraction to neurotransmitter release. The mechanism by which these channels select for Ca2+ over other cations is thought to involve multiple Ca2+-binding sites within the pore. Although the Ca2+ affinity and cation preference of these sites have been extensively investigated, the effect of voltage on these sites has not received the same attention. We used a neuronal preparation enriched for N-type calcium (CaV2.2) channels to investigate the effect of voltage on Ca2+ flux. We found that the EC50 for Ca2+ permeation increases from 13 mM at 0 mV to 240 mM at 60 mV, indicating that, during permeation, Ca2+ ions sense the electric field. These data were nicely reproduced using a three-binding-site step model. Using roscovitine to slow CaV2.2 channel deactivation, we extended these measurements to voltages <0 mV. Permeation was minimally affected at these hyperpolarized voltages, as was predicted by the model. As an independent test of voltage effects on permeation, we examined the Ca2+-Ba2+ anomalous mole fraction (MF) effect, which was both concentration and voltage dependent. However, the Ca2+-Ba2+ anomalous MF data could not be reproduced unless we added a fourth site to our model. Thus, Ca2+ permeation through CaV2.2 channels may require at least four Ca2+-binding sites. Finally, our results suggest that the high affinity of Ca2+ for the channel helps to enhance Ca2+ influx at depolarized voltages relative to other ions (e.g., Ba2+ or Na+), whereas the absence of voltage effects at negative potentials prevents Ca2+ from becoming a channel blocker. Both effects are needed to maximize Ca2+ influx over the voltages spanned by action potentials. 相似文献
17.
Pasham Parameshwar Reddy Vijeta Raghuram Johannes Hradsky Christina Spilker Asima Chakraborty Yogendra Sharma Marina Mikhaylova Michael R. Kreutz 《PloS one》2014,9(7)
Caldendrin, L- and S-CaBP1 are CaM-like Ca2+-sensors with different N-termini that arise from alternative splicing of the Caldendrin/CaBP1 gene and that appear to play an important role in neuronal Ca2+-signaling. In this paper we show that Caldendrin is abundantly present in brain while the shorter splice isoforms L- and S-CaBP1 are not detectable at the protein level. Caldendrin binds both Ca2+ and Mg2+ with a global Kd in the low µM range. Interestingly, the Mg2+-binding affinity is clearly higher than in S-CaBP1, suggesting that the extended N-terminus might influence Mg2+-binding of the first EF-hand. Further evidence for intra- and intermolecular interactions of Caldendrin came from gel-filtration, surface plasmon resonance, dynamic light scattering and FRET assays. Surprisingly, Caldendrin exhibits very little change in surface hydrophobicity and secondary as well as tertiary structure upon Ca2+-binding to Mg2+-saturated protein. Complex inter- and intramolecular interactions that are regulated by Ca2+-binding, high Mg2+- and low Ca2+-binding affinity, a rigid first EF-hand domain and little conformational change upon titration with Ca2+ of Mg2+-liganted protein suggest different modes of binding to target interactions as compared to classical neuronal Ca2+-sensors. 相似文献
18.
Previous studies showed that binding of water-soluble phosphatidylserine (C6PS) to bovine factor Xa (FXa) leads to Ca2+-dependent dimerization in solution. We report the effects of Ca2+, C6PS, and dimerization on the activity and structure of human and bovine FXa. Both human and bovine dimers are 106- to 107-fold less active toward prothrombin than the monomer, with the decrease being attributed mainly to a substantial decrease in kcat. Dimerization appears not to block the active site, since amidolytic activity toward a synthetic substrate is largely unaffected. Circular dichroism reveals a substantial change in tertiary or quaternary structure with a concomitant decrease in α-helix upon dimerization. Mass spectrometry identifies a lysine (K270) in the catalytic domain that appears to be buried at the dimer interface and is part of a synthetic peptide sequence reported to interfere with factor Va (FVa) binding. C6PS binding exposes K351 (part of a reported FVa binding region), K242 (adjacent to the catalytic triad), and K420 (part of a substrate exosite). We interpret our results to mean that C6PS-induced dimerization produces substantial conformational changes or domain rearrangements such that structural data on PS-activated FXa is required to understand the structure of the FXa dimer or the FXa-FVa complex. 相似文献
19.
V. N. Uversky S. E. Permyakov I. I. Senin A. M. Cherskaya S. V. Shulga-Morskoy D. V. Zinchenko A. M. Alekseev A. A. Zargarov V. M. Lipkin P. P. Philippov E. A. Permyakov 《Russian Journal of Bioorganic Chemistry》2000,26(3):152-156
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]. 相似文献
20.
A fluorescence method was adapted to investigate active ion transport in membrane preparations of the SR-Ca-ATPase. The styryl
dye RH421 previously used to investigate the Na,K-ATPase was replaced by an analogue, 2BITC, to obtain optimized fluorescence
changes upon substrate-induced partial reactions. Assuming changes of the local electric field to be the source of fluorescence
changes that are produced by uptake/release or by movement of ions inside the protein, 2BITC allowed the determination of
electrogenic partial reactions in the pump cycle. It was found that Ca2+ binding on the cytoplasmic and on the lumenal side of the pump is electrogenic while phosphorylation and conformational transition
showed only minor electrogenicity. Ca2+ equilibrium titration experiments at pH 7.2 in the two major conformations of the protein indicated cooperative binding of
two Ca2+ ions in state E1 with an apparent half-saturation concentration, K
M
of 600 nm. In state P-E2 two K
M
values, 5 μm and 2.2 mM, were determined and are in fair agreement with published data. From Ca2+ titrations in buffers with various pH and from pH titrations in P-E2, it could be demonstrated that H+ binding is electrogenic and that Ca2+ and H+ compete for the same binding site(s). Tharpsigargin-induced inhibition of the Ca-ATPase led to a state with a specific fluorescence
level comparable to that of state E1 with unoccupied ion sites, independent of the buffer composition.
Received: 21 September 1998/Revised: 18 December 1998 相似文献