The characterization for the binding of calcium and terbium to Euplotes octocarinatus centrin

Centrin is a member of the EF-hand superfamily that plays critical role in the centrosome duplication and separation. In the present paper, we characterized properties of metal ions binding to Euplotes octocarinatus centrin (EoCen) by fluorescence spectra and circular dichroism (CD) spectra. Changes of fluorescence spectra and alpha-helix contents of EoCen proved that Tb(3+) and Ca(2+) induced great conformational changes of EoCen resulting in exposing hydrophobic surfaces. At pH 7.4, Ca(2+) (and Tb(3+)) bond with EoCen at the ratio of 4:1. Equilibrium experiment indicated that Ca(2+) and Tb(3+) exhibited different binding capabilities for C- and N-terminal domains of protein. C-terminal domain bond with Ca(2+) or Tb(3+) approximately 100-fold more strongly than N-terminal. Aromatic residue-sensitized Tb(3+) energy transfer suggested that site IV bond to Tb(3+) or Ca(2+) more strongly than site III. Based on fluorescence titration curves, we reckoned the conditional binding constants of EoCen site IV quantitatively to be K(IV)=(1.23+/-0.51)x10(8)M(-1) and K(IV)=(6.82+/-0.33)x10(5)M(-1) with Tb(3+) and Ca(2+), respectively. Metal ions bond to EoCen in the order of IV>III>II, I.     

Spectral study on the binding of gadolinium ions with apoovotransferrin

The binding of Gd3+ ion to apoovotransferrin (apoOTf) was monitored by means of UV difference spectra in 0.01M Hepes, pH 7.4 at 25 degrees C. Used 2-p-toluidinylnaphthalene-6-sulfonate (TNS) as fluorescence probe the conformational changes of protein were studied while gadolinium ions bound to apoOTf. The results show that Gd3+ binding produces peaks at 244 and 294 nm that is the characteristic of binding at the apoOTf specific metal-binding sites. At 244 nm the molar absorptivity of Gd-apoOTf complex is (1.99+/-0.17)x10(4)cm(-1)M(-1). The apparent binding constants for the complexes of Gd3+ with apoovotransferrin are logK(1)=7.61+/-0.14 and logK(2)=4.96+/-0.26. A very large conformational change of apoovotransferrin appears when Gd3+ is bound to the N-terminal binding site. When Gd3+ is bound to C-terminal binding site there is less conformational change.     

The spectral studies on the effect of Glu 101 to the metal binding characteristic of Euplotes octocarinatus centrin

Glu is highly conserved as the first amino acid of E-helix of the EF-hand protein. In this paper, Glu 101, the first amino acid of E-helix of the third EF-hand motif in Euplotes octocarinatus centrin (EoCen) was mutated to be Lys by the method of site direct mutation. Tb3+ and TNS were used as fluorescence probes in the study of the effect of this mutation to the metal binding characteristic of EoCen by fluorescence spectra. Results indicate that compared with EoCen, the mutation protein (E101K) displays a different Tb3+ binding characteristic and an increased hydrophobic exposure surface. Polyacrylamide gels electrophoresis indicated that the electrophoretic mobilities of EoCen and E101K are distinctly different. It can be deduced that the conformation of EoCen has been altered by this mutation. The general conditional binding constant of Tb3+ to the three loops of EF-hand sites I-III in E101K was calculated to be (5.64+/-0.57)x10(5)M(-1) according to the modified equation of the single binding process.     

Room temperature and shock tube study of the reaction HCO+O2 using the photolysis of glyoxal as an efficient HCO source

The rate of the reaction 1, HCO+O2-->HO2+CO, has been determined (i) at room temperature using a slow flow reactor setup (20 mbarH2+HCO+CO, into additional HCO radicals. The rate constants of reaction 4 were determined from unperturbed photolysis experiments to be k4(295 K)=(3.6+/-0.3)x10(10) cm3 mol-1 s-1 and k4(769-1107 K)=5.4x10(13)exp(-18 kJ mol-1/RT) cm3 mol-1 s-1(Delta log k4=+/-0.12).     

尖吻蝮蛇毒抗凝血因子Ⅱ的稀土离子荧光探针研究

尖吻蝮蛇毒抗凝血因子(ACF)分子中有两个钙离子结合位点,钙离子对ACF的内源荧光有增强作用,稀土离子(Nd³⁺,Sm³⁺,Eu³⁺,Gd³⁺和Tb³⁺)能取代ACF分子中的钙离子,并对ACF的内源荧光有不同程度的猝灭作用,其中Tb³⁺接受ACF分子中Trp残基传递的能量后,特征荧光增强.稀土离子与ACF荧光滴定表明,ACF分子中有两个稀土离子结合位点,稀土离子和钙离子在ACF分子中两个结合部位是共同的竞争结合部位.ACF与不同稀土离子之间有相近的表观结合常数K₁或K₂.Tb³⁺与RE³⁺(RE=Nd,Sm,Eu或Gd)间线性自由能关系表明,稀土离子与ACF结合时,没有明显的空间效应.ACF分子中的两个结合位点在结构上都有较大的柔性,这种结构柔性为钙离子在ACF与活化凝血因子X的结合反应中起到的促进作用提供了结构基础.     

Diazacoronand linked beta-cyclodextrin dimer complexes of Brilliant Yellow tetraanion and their sodium(I) analogues

Complexation of the Brilliant Yellow tetraanion, 3(4-), by two new diazacoronand linked beta-cyclodextrin (beta CD) dimers 4,13-bis(2-(6A-deoxy-beta-cyclodextrin-6A-yl)aminooctylamidomethyl- and 4,13-bis(8-(6A-deoxy-beta-cyclodextrin-6A-yl)aminooctylamidomethyl)-4,13- diaza-1,7,10-trioxacyclopentadecane, 1 and 2, respectively, has been studied in aqueous solution. UV-visible spectrophotometric studies at 298.2 K, pH 10.0 and I = 0.10 mol dm-3 (NEt4ClO4) yielded complexation constants for the complexes 1 x 3(4-) and 2 x 3(4-), K1 = (1.08 +/- 0.01) x 10(5) and (6.21 +/- 0.08) x 10(3) dm3 mol-1, respectively. Similar studies at 298.2 K, pH 10.0 and I = 0.10 mol dm-3 (NaClO4) yielded K3 = (4.63 +/- 0.09) x 10(5) and (3.38 +/- 0.05) x 10(4) dm3 mol-1 for the complexation of 3(4-) by Na+ x 1 and Na+ x 2 to give Na+ x 1 x 3(4-) and Na+ x 2 x 3(4-), respectively. Potentiometric studies of the complexation of Na+ by 1 and 2 by the diazacoronand component of the linkers to give Na+ x 1 and Na+ x 2 yielded K2 = (2.00 +/- 0.05) x 10(3) and (1.8 +/- 0.05) x 10(3) dm3 mol-1, respectively, at 298.2 K and I = 0.10 mol dm-3(NEt4ClO4). For complexation of Na+ by 1 x 3(4-) and 2 x 3(4-) to give Na+ x 1 x 3(4-) and Na+ x 2 x 3(4-) K2K3/K1 = K4 = 8.6 x 10(2) and 9.8 x 10(3) dm3 mol-1, respectively. The pKaS of 1H4(4+) are 7.63 +/- 0.01, 6.84 +/- 0.02, 5.51 +/- 0.04 and 4.98 +/- 0.03, and those of 2H4(4+) are 8.67 +/- 0.02, 8.11 +/- 0.02, 6.06 +/- 0.02 and 5.14 +/- 0.05. The larger magnitude of K1 for 1 by comparison with K1 for 2 is attributed to the octamethylene linkers of 2 competing with 3(4-) for occupancy of the annuli of the beta CD entities while the competitive ability of the dimethylene linkers of 1 is less. A similar argument applies to the relative magnitudes of K3 for Na+ x 1 and Na+ x 2. Increased electrostatic attraction probably accounts for K3 > K1 for Na+ x 1 x 3(4-) and 1 x 3(4-) and for Na+ x 2 x 3(4-) and 2 x 3(4-). The lesser magnitudes of K2 and K4 for Na+ x 1 and Na+ x 1 x 3(4-) compared with those for Na+ x 2 and Na+ x 2 x 3(4-) are attributed to the octamethylene linkers of 2 producing a more hydrophobic environment for the diazacoronand than that produced by the dimethylene linkers of 1. 1H NMR spectroscopic studies and the syntheses of 1 and 2 are described.     

钙(Ⅱ)对尖吻蝮蛇毒抗凝血因子Ⅰ的抗凝血活性的影响

皖南尖吻蝮蛇毒抗凝血因子Ⅰ(ACFⅠ)不具有酶的活性,通过与活化凝血因子X(FXa)结合成1l的复合物来延长凝血时间。用高效液相色谱分析方法,发现ACFⅠ与FXa的结合反应依赖于Ca     

Dissociative recombination of OPCl+ and OPCl2+: pushing the upper mass limit at CRYRING

The dissociative recombination of OPCl+ and OPCl2+ has been studied at the storage ring CRYRING. The rate constants as a function of electron temperature have been derived to be 7.63 x 10(-7)(Te/300)(-0.89) and >1.2 x 10(-6)(Te/300)(-1.22) cm3s(-1), respectively. The lower limit quoted for the latter rate constant reflects the experimental inability to detect all of the reaction products. The branching fractions from the reaction have been measured for OPCl+ at approximately 0 eV interaction energy and are determined to be N(O+P+Cl)=(16+/-7)%, N(O+PCl)=(16+/-3)% and N(OP+Cl)=(68+/-5)%. These values have been obtained assuming that the rearrangement channel forming P+ClO is negligible, and ab initio calculations using GAUSSIAN03 are presented for the ion structures and energetics to support such an assumption. Finally, the limitations to using heavy ion storage rings such as CRYRING for studies into the dissociative recombination of large singly charged molecular ions are discussed.     

Rate coefficients for the reaction of OH with (E)-2-pentenal, (E)-2-hexenal, and (E)-2-heptenal

Rate coefficients for the gas-phase reaction of the OH radical with (E)-2-pentenal (CH(3)CH(2)CH[double bond]CHCHO), (E)-2-hexenal (CH(3)(CH(2))(2)CH[double bond]CHCHO), and (E)-2-heptenal (CH(3)(CH(2))(3)CH[double bond]CHCHO), a series of unsaturated aldehydes, over the temperature range 244-374 K at pressures between 23 and 150 Torr (He, N(2)) are reported. Rate coefficients were measured under pseudo-first-order conditions in OH with OH radicals produced via pulsed laser photolysis of HNO(3) or H(2)O(2) at 248 nm and detected by pulsed laser-induced fluorescence. The rate coefficients were independent of pressure and the room temperature rate coefficients and Arrhenius expressions obtained are (cm(3) molecule(-1) s(-1) units): k(1)(297 K)=(4.3 +/- 0.6)x 10(-11), k(1)(T)=(7.9 +/- 1.2)x 10(-12) exp[(510 +/- 20)/T]; k(2)(297 K)=(4.4 +/- 0.5)x 10(-11), k(2)(T)=(7.5 +/- 1.1)x 10(-12) exp[(520 +/- 30)/T]; and k(3)(297 K)=(4.4 +/- 0.7)x 10(-11), k(3)(T)=(9.7 +/- 1.5)x 10(-12) exp[(450 +/- 20)/T] for (E)-2-pentenal, (E)-2-hexenal and (E)-2-heptenal, respectively. The quoted uncertainties are 2sigma(95% confidence level) and include estimated systematic errors. Rate coefficients are compared with previously published room temperature values and the discrepancies are discussed. The atmospheric degradation of unsaturated aldehydes is also discussed.     

金属离子对齐多夫定与牛血清白蛋白结合作用的影响

用荧光光谱法和紫外分光光度法研究了水溶液(Tris-HCl缓冲溶液, pH 7.1)中齐多夫定(ZDV)与牛血清白蛋白(BSA)的结合作用及三种金属离子(Cu2+, Mg2+, Zn2+)对其的影响. 结果表明: 齐多夫定及金属离子均导致BSA的内源荧光猝灭, 猝灭机制均为静态猝灭; 齐多夫定与BSA间存在较强结合作用, 热力学参数△H和△S分别为-10.2 kJ·mol-1和77.5 J·mol-1·K-1 (298 K), 表明其结合力以静电作用力为主; 298 K下结合常数、结合位点数和结合距离分别为6.92×105 L·mol-1、1.18和2.28 nm; 温度升高结合常数和结合位点数减小. 三种金属离子均导致ZDV与BSA的结合常数减小, 结合距离增大.     

Ferrocenyl-bearing cyclopseudopeptides as redox-switchable cation receptors

A family of ferrocenyl-bearing cyclopseudopeptides (1-10) designed for redox-switchable receptors of cations was synthesized. Circular dichroism (CD) and cyclic valtammetry (CV) studies of cation binding properties in both the reduced (K1) and oxidized (K2) forms revealed that the binding preference is mainly governed by the charges and radius of the guest cation as well as by the suitability of the host to accommodate the guest. Particularly worth mentioning is the fact that some synthesized cyclopseudopeptides showed high binding affinity and selectivity toward alkaline-earth ions. For example, the K1 of compound 2 binding with Ca2+ is 4.37 x 10(6) mol(-1) x L and its Ca2+/K+ selectivity is 3.1 x 10(5):1, both values are much greater than those of an excellent natural ionophore, valinomycin (1 x 10(5) mol(-1) x L and 0.33:1, respectively). The linear relationship between the shifts of half-wave potentials (deltaE(1/2)) and the radius/charge [r/(+)] ratios suggests that the sensitivity of electrochemical responses to cation complexation be dominated by repulsion factors between the redox center and the incoming cation guest.     

A kinetic study of Ca-containing ions reacting with O, O2, CO2 and H2O: implications for calcium ion chemistry in the upper atmosphere

A series of gas-phase reactions involving molecular Ca-containing ions was studied by the pulsed laser ablation of a calcite target to produce Ca(+) in a fast flow of He, followed by the addition of reagents downstream and detection of ions by quadrupole mass spectrometry. Most of the reactions that were studied are important for describing the chemistry of meteor-ablated calcium in the earth's upper atmosphere. The following rate coefficients were measured: k(CaO(+) + O --> Ca(+) + O(2)) = (4.2 +/- 2.8) x 10(-11) at 197 K and (6.3 +/- 3.0) x 10(-11) at 294 K; k(CaO(+) + CO --> Ca(+) + CO(2), 294 K) = (2.8 +/- 1.5) x 10(-10); k(Ca(+).CO(2) + O(2) --> CaO(2)(+) + CO(2), 294 K) = (1.2 +/- 0.5) x10(-10); k(Ca(+).CO(2) + H(2)O --> Ca(+).H(2)O + CO(2)) = (13.0 +/- 4.0) x 10(-10); and k(Ca(+).H(2)O + O(2) --> CaO(2)(+) + H(2)O, 294 K) = (4.0 +/- 2.5) x 10(-10) cm(3) molecule(-1) s(-1). The quoted uncertainties are a combination of the 1sigma standard errors in the kinetic data and the systematic errors in the models used to extract the rate coefficients. Rate coefficients were also obtained for the following recombination (also termed association) reactions in He bath gas: k(Ca(+).CO(2) + CO(2) --> Ca(+).(CO(2))(2), 294 K) = (2.6 +/- 1.0) x 10(-29); k(Ca(+).H(2)O + H(2)O --> Ca(+).(H(2)O)(2)) = (1.6 +/- 1.1) x 10(-27); and k(CaO(2)(+) + O(2) --> CaO(2)(+).O(2)) < 1 x 10(-31) cm(6) molecule(-2) s(-1). These recombination rate coefficients, as well as those for the ligand-switching reactions listed above, were then interpreted using a combination of high level quantum chemistry calculations and RRKM theory using an inverse Laplace transform solution of the master equation. The surprisingly slow reaction between CaO(+) and O was explained using quantum chemistry calculations on the lowest (2)A', (2)A' and (4)A' potential energy surfaces. These calculations indicate that reaction mostly occurs on the (2)A' surface, leading to production of Ca(+)((2)S) + O(2)((1)Delta(g)). The importance of this reaction for controlling the lifetime of Ca(+) in the upper mesosphere and lower thermosphere is then discussed.     

Relative and absolute kinetic studies of 2-butanol and related alcohols with tropospheric Cl atoms

A newly constructed chamber/Fourier transform infrared system was used to determine the relative rate coefficient, k(i), for the gas-phase reaction of Cl atoms with 2-butanol (k(1)), 2-methyl-2-butanol (k(2)), 3-methyl-2-butanol (k(3)), 2,3-dimethyl-2-butanol (k(4)) and 2-pentanol (k(5)). Experiments were performed at (298 +/- 2) K, in 740 Torr total pressure of synthetic air, and the measured rate coefficients were, in cm(3) molecule(-1) s(-1) units (+/-2sigma): k(1)=(1.32 +/- 0.14) x 10(-10), k(2)=(7.0 +/- 2.2) x 10(-11), k(3)=(1.17 +/- 0.14) x 10(-10), k(4)=(1.03 +/- 0.17) x 10(-10) and k(5)=(2.18 +/- 0.36) x 10(-10), respectively. Also, all the above rate coefficients (except for 2-pentanol) were investigated as a function of temperature (267-384 K) by pulsed laser photolysis-resonance fluorescence (PLP-RF). The obtained kinetic data were used to derive the Arrhenius expressions: k(1)(T)=(6.16 +/- 0.58) x 10(-11)exp[(174 +/- 58)/T], k(2)(T)=(2.48 +/- 0.17) x 10(-11)exp[(328 +/- 42)/T], k(3)(T)=(6.29 +/- 0.57) x 10(-11)exp[(192 +/- 56)/T], and k(4)(T)=(4.80 +/- 0.43) x 10(-11)exp[(221 +/- 56)/T](in units of cm(3) molecule(-1) s(-1) and +/-sigma). Results and mechanism are discussed and compared with the reported reactivity with OH radicals. Some atmospheric implications derived from this study are also reported.     

ESR and optical study of Cu2+ doped calcium malonate dihydrate

The ESR study of Cu(2+) doped calcium malonate dihydrate has been done at room temperature. Four magnetically in-equivalent sites for Cu(2+) have been observed. The spin-Hamiltonian parameters evaluated with the fitting of spectra to rhombic symmetry crystalline field are for Cu(2+) site (I): g(x)=2.0963+/-0.0002, g(y)=2.1316+/-0.0002, g(z)=2.4137+/-0.0002, A(x)=(32+/-2)x10(-4)cm(-1), A(y)=(34+/-2)x10(-4)cm(-1), A(z)=(49+/-2)x10(-4)cm(-1), for site (II): g(x)=2.0668+/-0.0002, g(y)=2.0800+/-0.0002, g(z)=2.3561+/-0.0002, A(x)=(34+/-2)x10(-4)cm(-1), A(y)=(36+/-2)x10(-4)cm(-1), A(z)=(51+/-2)x10(-4)cm(-1), for site (III): g(x)=2.0438+/-0.0002, g(y)=2.0623+/-0.0002, g(z)=2.2821+/-0.0002, A(x)=(34+/-2)x10(-4)cm(-1), A(y)=(36+/-2)x10(-4)cm(-1), A(z)=(53+/-2)x10(-4)cm(-1), and for site (IV): g(x)=2.0063+/-0.0002, g(y)=2.0241+/-0.0002, g(z)=2.2357+/-0.0002, A(x)=(35+/-2)x10(-4)cm(-1), A(y)=(37+/-2)x10(-4)cm(-1), A(z)=(54+/-2)x10(-4)cm(-1). The ground state wave function of Cu(2+) has also been determined. The g-anisotropy has been estimated and compared with the experimental value. Further with the help of optical study the nature of bonding of metal ion with different ligands in the complex has been discussed.     

Novel chiral N4S2- and N6S3-donor macrocyclic ligands: synthesis,protonation constants,metal-ion binding and asymmetric catalysis in the Henry reaction

New hydrophobic chiral macrocyclic ligands L1-L3 with chiral diamino and thiophene moieties have been synthesized by the Schiff base condensation approach. Protonation constants of L1 and L2 were determined by potentiometry titration. Metal-ion binding experiments exhibited that L1 and L3 are pronounced in selective recognition, Ag+, Cu2+ and Ca2+ ions among the surveyed metal ions (Cu2+, Co2+, Ni2+, Zn2+, Cd2+, Pb2+, Ag+, Li+, Na+, K+, and Ca2+). L1 was found to spectroscopically detect the presence of Cu2+ and Ca2+ to function as a multiple readout sensor. The detection limit for Ca2+ ions was found to be 9.8 x 10(-5) M in CH2Cl2-MeOH solution. The trimeric chiral ligand L3 has been shown to be an efficient auxiliary in a Zn(II)-mediated enantioselective Henry reaction.     

Probing metal binding in the 8-17 DNAzyme by TbIII luminescence spectroscopy

Metal-dependent cleavage activities of the 8-17 DNAzyme were found to be inhibited by Tb(III) ions, and the apparent inhibition constant in the presence of 100 microM of Zn(II) was measured to be 3.3+/-0.3 microM. The apparent inhibition constants increased linearly with increasing Zn(II) concentration, and the inhibition effect could be fully rescued with addition of active metal ions, indicating that Tb(III) is a competitive inhibitor and that the effect is completely reversible. The sensitized Tb(III) luminescence at 543 nm was dramatically enhanced when Tb(III) was added to the DNAzyme-substrate complex. With an inactive DNAzyme in which the GT wobble pair was replaced with a GC Watson-Crick base pair, the luminescence enhancement was slightly decreased. In addition, when the DNAzyme strand was replaced with a complete complementary strand to the substrate, no significant luminescence enhancement was observed. These observations suggest that Tb(III) may bind to an unpaired region of the DNAzyme, with the GT wobble pair playing a role. Luminescence lifetime measurements in D(2)O and H(2)O suggested that Tb(III) bound to DNAzyme is coordinated by 6.7+/-0.2 water molecules and two or three functional groups from the DNAzyme. Divalent metal ions competed for the Tb(III) binding site(s) in the order Co(II)>Zn(II)>Mn(II)>Pb(II)>Ca(II) approximately Mg(II). This order closely follows the order of DNAzyme activity, with the exception of Pb(II). These results indicate that Pb(II), the most active metal ion, competes for Tb(III) binding differently from other metal ions such as Zn(II), suggesting that Pb(II) may bind to a different site from that for the other metal ions including Zn(II) and Tb(III).     

Kinetics of manganese(III) acetate in acetic acid: generation of Mn(III) with Co(III), Ce(IV), and dibromide radicals; reactions of Mn(III) with Mn(II), Co(II), hydrogen bromide,and alkali bromides

The reaction of cobalt(III) acetate with excess manganese(II) acetate in acetic acid occurs in two stages, since the two forms Co(IIIc) and Co(IIIs) are not rapidly equilibrated and thus react independently. The rate constants at 24.5 degrees C are kc = 37.1 +/- 0.6 L mol-1 s-1 and ks = 6.8 +/- 0.2 L mol-1 s-1 at 24.5 degrees C in glacial acetic acid. The Mn(III) produced forms a dinuclear complex with the excess of Mn(II). This was studied independently and is characterized by the rate constant (3.43 +/- 0.01) x 10(2) L mol-1 s-1 at 24.5 degrees C. A similar interaction between Mn(III) and Co(II) is substantially slower, with k = (3.73 +/- 0.05) x 10(-1) L mol-1 s-1 at 24.5 degrees C. Mn(II) is also oxidized by Ce(IV), according to the rate law -d[Ce(IV)]/dt = k[Mn(II)]2[Ce(IV)], where k = (6.0 +/- 0.2) x 10(4) L2 mol-2 s-1. The reaction between Mn(II) and HBr2., believed to be involved in the mechanism by which Mn(III) oxidizes HBr, was studied by laser photolysis; the rate constant is (1.48 +/- 0.04) x 10(8) L mol-1 s-1 at approximately 23 degrees C in HOAc. Oxidation of Co(II) by HBr2. has the rate constant (3.0 +/- 0.1) x 10(7) L mol-1 s-1. The oxidation of HBr by Mn(III) is second order with respect to [HBr]; k = (4.10 +/- 0.08) x 10(5) L2 mol-2 s-1 at 4.5 degrees C in 10% aqueous HOAc. Similar reactions with alkali metal bromides were studied; their rate constants are 17-23 times smaller. This noncomplementary reaction is believed to follow that rate law so that HBr2. and not Br. (higher in Gibbs energy by 0.3 V) can serve as the intermediate. The analysis of the reaction steps then requires that the oxidation of HBr2. to Br2 by Mn(III) be diffusion controlled, which is consistent with the driving force and seemingly minor reorganization.     

Probing site-specific calmodulin calcium and lanthanide affinity by grafting

Ca2+ binding is essential for the biological functions of calmodulin (CaM) as a trigger/sensor protein to regulate many biological processes in the Ca2+ -signaling cascade. A challenge in understanding the mechanism of Ca2+ signaling is to obtain site-specific information about the Ca2+ binding properties of individual Ca2+ -binding sites of EF-hand proteins, especially for CaM. In this paper, we report the first estimation of the intrinsic Ca2+ affinities of the four EF-hand loops of calmoduin (I-IV) by individually grafting into the domain 1 of CD2. Taking advantage of the Trp residues in the host protein, we first determined metal-binding affinities for Tb3+, Ca2+, and La3+ for all four grafted EF-loops using Tb3+ aromatic resonance energy transfer. EF-loop I exhibits the strongest binding affinity for Ca2+, La3+, and Tb3+, while EF-loop IV has the weakest metal-binding affinity. EF-loops I-IV of CaM have dissociation constants for Ca2+ of 34, 245, 185, and 814 microM, respectively, with the order I > III approximately equal to II > IV. These findings support a charge-ligand-balanced model in which both the number of negatively charged ligand residues and the balanced electrostatic dentate-dentate repulsion by the adjacent charged residues are two major determinants for the relative Ca2+ -binding affinities of EF-loops in CaM. Our grafting method provides a new strategy to obtain site-specific Ca2+ binding properties and a better estimation of the cooperativity and conformational change contributions of coupled EF-hand proteins.     

Kinetics of the reactions of CH2Br and CH2I radicals with molecular oxygen at atmospheric temperatures

The kinetics of the reactions of CH2Br and CH2I radicals with O2 have been studied in direct measurements using a tubular flow reactor coupled to a photoionization mass spectrometer. The radicals have been homogeneously generated by pulsed laser photolysis of appropriate precursors at 193 or 248 nm. Decays of radical concentrations have been monitored in time-resolved measurements to obtain the reaction rate coefficients under pseudo-first-order conditions with the amount of O2 being in large excess over radical concentrations. No buffer gas density dependence was observed for the CH2I + O2 reaction in the range 0.2-15 x 10(17) cm(-3) of He at 298 K. In this same density range the CH2Br + O2 reaction was obtained to be in the third-body and fall-off area. Measured bimolecular rate coefficient of the CH2I + O2 reaction is found to depend on temperature as k(CH2I + O2)=(1.39 +/- 0.01)x 10(-12)(T/300 K)(-1.55 +/- 0.06) cm3 s(-1)(220-450 K). Obtained primary products of this reaction are I atom and IO radical and the yield of I-atom is significant. The rate coefficient and temperature dependence of the CH2Br + O2 reaction in the third-body region is k(CH2Br + O2+ He)=(1.2 +/- 0.2)x 10(-30)(T/300 K)(-4.8 +/- 0.3) cm6 s(-1)(241-363 K), which was obtained by fitting the complete data set simultaneously to a Troe expression with the F(cent) value of 0.4. Estimated overall uncertainties in the measured reaction rate coefficients are about +/-25%.     

Influence of metal cations on the intramolecular hydrogen-bonding network and pKa in phosphorylated compounds

The binding of the most common metal cations of cytoplasm (Li+, Na+, K+, Mg2+ and Ca2+) to a model molecule having an intramolecular hydrogen-bonding network, myo-inositol-2-monophosphate, was studied using first principles. A strong correlation between the conformation of metal inositol phosphate complexes with the type of metal cation, degree of deprotonation state, and the surrounding environment has been observed. On the basis of the hydrogen-bonding network analysis of the cation-phosphate complexes (Mn+-Ins(2)P1), the alkali cations show little effect on the conformational preference while the conformational preference for the binding of the alkaline earth cations is pH-dependent and solvent-dependent. For example, these calculations predict that Mg2+-Ins(2)P1(0) and Mg2+-Ins(2)P1(2-) favor the 1a/5e form while Mg2+-Ins(2)P1(1-) favors the 5a/1e conformation. The Ca2+-Ins(2)P1(2-) complex prefers the 1a/5e conformation in the gas phase and in a nonpolar protein environment, but inverts to the 5a/1e conformation upon entering the polar aqueous phase. The binding affinities of the cations and the pK(a) values for the cation-phosphate complexes are derived from thermodynamical analysis.