A whole-cell assay for the high throughput screening of calmodulin antagonists

Cell-based screening systems for pharmaceuticals are desired over molecular biosensing systems because of the information they provide on toxicity and bioavailability. However, the majority of sensing systems developed are molecular biosensing type screening systems and cannot be easily adapted to cell-based screening. In this study, we demonstrate that protein-based molecular sensing systems that employ a fluorescent protein as a signal transducer are amenable to cell-based sensing by expressing the protein molecular sensing system in the cell and employing these cells for screening of desired molecules. To achieve this, we expressed a molecular sensing system based on the fusion protein of calmodulin (CaM) and enhanced green fluorescent protein (EGFP) in bacterial cells, and utilized these cells for the screening of CaM antagonists. In the presence of Ca²⁺, CaM undergoes a conformational change exposing a hydrophobic pocket that interacts with CaM-binding proteins, peptides, and drugs. This conformational change induced in CaM leads to a change in the microenvironment of EGFP, resulting in a change in its fluorescence intensity. The observed change in fluorescence intensity of EGFP can be correlated to the concentration of the analyte present in the sample. Dose-response curves for various tricyclic antidepressants were generated using cells containing CaM-EGFP fusion protein. Additionally, we demonstrate the versatility of our system for studying protein-protein interactions by using cells to study the binding of a peptide to CaM. The study showed that the CaM-EGFP fusion protein within the intact cells responds similarly to that of the isolated fusion protein, hence eliminating the need for any isolation and purification steps. We have demonstrated that this system can be used for the rapid screening of various CaM antagonists that are potential antipsychotic drugs.     

Molecular dynamics study of the influence of calcium ions on the conformation of gelsolin S2 domain

Gelsolin is an actin-severing protein whose action is promoted by Ca²⁺ ions and inhibited by binding to lipid phosphoinositides incorporated in the inner leaflet of the plasma membrane inner lipid bilayer. In this study, we carried out molecular dynamics (MD) simulations to investigate the influence of calcium cations on the conformation of gelsolin S2 domain. First, gelsolin S2 domain taken from the crystal structure of apo-gelsolin (PDB code: 1D0N) was subjected to three 1100 ps MD simulations in a periodic water box with the 5.0 force field at T=298 K. In the first simulation (S2_Ca²⁺) excess concentration of Ca²⁺ was applied, in the second one (S2_phys) the concentration of Ca²⁺ ions was physiological and in the third one (S2_w) no Ca²⁺ ions were added. The results of MD simulations showed high conformational flexibility of the N-terminal part of the S2 domain. S2_w deviated from the starting structure considerably more that S2_phys and S2_Ca²⁺ suggesting that Ca²⁺ ions stabilize the conformation of the S2 domain of gelsolin.     

钙离子和镁离子浓度变化对磷脂酰乙醇胺-磷脂酰甘油双分子层膜的影响

Ca²⁺ and Mg²⁺ ions are the main divalent cations in living cells and play vital roles in the structure and function of biological membranes. To date, the differences in the effects of these two ions on the Escherichia coli (E. coli) inner membrane at various concentrations remain unknown. Here, the effects of Ca²⁺ and Mg²⁺ ions on a mixed lipid bilayer composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) in a 3 : 1 ratio (mol/mol), which mimics the E. coli inner membrane, were quantitatively differentiated at different concentrations by dynamic light scattering (DLS), zeta potential measurements and all-atom molecular dynamics (AA-MD) simulations. The DLS results demonstrated that the POPE/POPG liposomes were homogeneous and monodisperse in solutions with Ca²⁺ or Mg²⁺ ion concentrations of 0 and 1 mmol∙L^-1. As the Ca²⁺ or Mg²⁺ ion concentration was increased to 5-100 mmol∙L^-1, lipid aggregation or the fusion of unilamellar liposomes occurred in the ion solutions. The zeta potential measurements showed that both the Ca²⁺ and Mg²⁺ ions had overcharging effects on the negatively charged POPE/POPG liposomes. The AA-MD simulation results indicated that the Ca²⁺ ions irreversibly adsorbed on the membranes when the simulation time was longer than 100 ns, while the Mg²⁺ ions were observed to dynamically adsorb on and desorb from the membranes at various concentrations. These results are consistent with the DLS and zeta potential experiments. The average numbers of Ca²⁺ and Mg²⁺ ions in the first coordination shell of the oxygen atoms of the phosphate, carbonyl and hydroxyl groups of POPE and POPG (i.e., the first coordination numbers) in the pure membrane and membranes containing 5 and 100 mmol∙L^-1 ions were calculated from the radial distribution functions. The results indicated that the primary binding site of these two ions on POPE and POPG at the concentrations studied was the negatively charged phosphate group. Thus, these results might explain the overcharging effects of both the Ca²⁺ and Mg²⁺ ions on the POPE/POPG liposomes. Moreover, as the Ca²⁺ concentration increased, the area per lipid of the lipid bilayers decreased, and the membrane thickness increased, while the Mg²⁺ ions had negligible effects on these membrane parameters. In addition, these ions had different effects on the orientation of the lipid head groups. These simulation results may be used to provide the possible explanations for the differences between Ca²⁺ and Mg²⁺ ions in DLS and zeta potential measurements at the atomic level. The experimental results and MD simulations provide insight into various biological processes regulated by divalent cations, such as membrane fusion.     

Surface properties of plasma membrane vesicles isolated from melon (Cucumus melo L.) root cells differing in salinity tolerance

The hypotheses that genotypic differences in salinity tolerance may result from (i) differences in global surface charge density or (ii) from differences in global Ca²⁺ binding were tested. An attempt was made to correlate the differing salinity tolerance of four melon cultivars with surface properties of vesicles extracted from the plasma membrane (PM) of their root cells. Surface characterization involved measurements of electrophoretic mobility and sorption of ⁴⁵Ca²⁺ to the vesicles in the presence of varying concentrations of Ca²⁺, Na⁺ and Mg²⁺. Irrespective of salinity tolerance, vesicles from the four cultivars yielded similar ζ potentials under similar conditions, indicating similar global surface charge densities. Sorption studies with vesicles from two cultivars differing in salinity tolerance predicted independently this result of equal surface charge density. The estimated global binding affinities of Ca²⁺, Na⁺ and Mg²⁺ to the PM of both cultivars were the same with binding coefficients of 50, 0.8 and 9 M⁻¹, respectively. Consequently, the hypotheses enumerated above to interpret genotypic differences in salinity toxicity are rejected. However, vesicles from the salt-resistant strain sorbed 19% more Ca²⁺ per given amount of protein in the membrane, indicating the existence of a larger number of negatively charged surface sites per given amount of protein and a smaller amount of protein per given area of membrane. Genotypic differences in site-specific Ca²⁺-binding affinity (e.g. at ion channels) remain a viable hypothesis for genotypic differences in salinity tolerance.     

BSA与羟磷灰石相互吸附的FTIR-ATR光谱

采用傅里叶变换红外衰减全反射(FTIR-ATR)光谱法对牛血清白蛋白(BSA)在羟磷灰石(HA)[Ca₁₀(OH)₂(PO₄)₆]表面不同时间的相互吸附作用进行了表征。在BSA溶液作用下,羟磷灰石表面的Ca²⁺、PO₄^3-和OH^-离子初始的溶解和再沉淀使得BSA与HA相互作用层层叠加,在HA表面形成从表层到次表层分子都包含有吸附的BSA的覆盖层,从而加深两者之间的相互作用。经红外差谱法处理过的相关ATR数据表明, BSA与HA之间的相互作用是快速的,并随时间变化进一步加强;来自HA上PO₄^3-的P=O基团对蛋白质肽键的酰胺II带(―CNH)、多肽链的甲基(―PO₃)和亚甲基(―CH₂)上氢的吸附作用要比P―O快速而且强烈。Ca²⁺在该吸附过程中起了极其重要的作用,其快速与蛋白质肽键的羰基氧发生作用,并诱导该蛋白质二级结构由β-折叠向α-螺旋和β-转角构象转变;伴随着这一构象变化,蛋白质多肽链上大多数肽键的―C=O和H―N―活性基团从链间氢键交联中释放出来,带动众多的氢分别参与同HA表面的Ca²⁺、PO43-和OH^-离子的相互吸附作用,并牢牢地结合于HA表面;这对硬组织的再生起着重要作用,促进了HA的生物矿化过程。     

氧化镥中杂质对铈掺杂硅酸镥多晶粉体光谱性质影响

氧化镥中杂质元素对其分离制备工艺、生产成本及镥基硅酸盐闪烁晶体的性能有很大影响,但杂质元素对晶体的性质影响研究较为有限,有待进一步完善。 本文采用高温固相法制备了Lu₂O₃分离过程中关键杂质元素Yb³⁺和Ca²⁺共掺杂Lu₂SiO₅∶Ce多晶粉体,研究了Yb³⁺或Ca²⁺含量对多晶粉体光谱性质的影响,结果表明:Yb³⁺或Ca²⁺共掺并未改变发射光谱的形状和位置,随着杂质元素摩尔分数的增加,光谱强度和荧光寿命逐渐降低。     

Density functional theory assessment of the thermal degradation of diclofenac and its calcium and iron complexes

Thermogravimetric analyses of diclofenac sodium, its Ca²⁺ and Fe³⁺ complexes manifested a decreasing trend of the onset decomposition temperatures at which these compounds dissociated. The drop in the temperature was metal ion dependent; the sodium salt showed thermal stability up to 245 °C, whereas the complexes started their degradation processes at temperatures starting from 90 °C. While G* for the cleavage of the acetate moiety in the sodium salt was 63.76 kJmol⁻¹, it was 82.06 and 140.57 kJmol⁻¹ in the cases of Ca²⁺ and Fe³⁺, respectively. However, their complete fusion took place at 187.65, 150.34 and 98.77 °C, respectively, displaying a reversed trend which is probably indicative of some catalytic part on the binding metals.

Using the Gaussian 98 W package of programs, ab initio molecular orbital treatments were applied to diclofenac and its Ca²⁺ and Fe³⁺ metal complexes to study their electronic structure at the atomic level. The thermochemistry of diclofenac sodium was followed through the TG fragmentation peak temperatures using the density functional theory calculations at the 6-31G(d) basis set level. The FT-IR data were in good agreement with the theoretically calculated values.

Single point calculations at the B3LYP/ 6-311G(d) level of theory, were used to compare the geometric features, energies and dipole moments of these compounds to detect the effect of the binding metal ions on the thermal dissociation of their diclofenac complexes.     

Comparison of negative and positive ion electrospray ionization mass spectra of calmodulin and its complex with trifluoperazine

The protein calmodulin (apoCaM) undergoes a conformational change when it binds calcium. This structure of the protein (Ca4CaM) is a dumbbell-shaped molecule that undergoes a further profound conformational change on binding of the antipsychotic drug trifluoperazine (TFP). Experimental conditions were developed to prepare samples of apoCaM, Ca4CaM and Ca4CaM/TFP that were substantially free of sodium. The effects of the conformational changes of calmodulin on the charge-state distributions observed in positive ion and negative ion electrospray ionization (ESI) mass spectra were examined. Conversion of apoCaM into Ca4CaM was concomitant with a change in the negative ion ESI mass spectrum whereby the 16- ion was the most abundant ion observed for the apo form and the 8- ion was the most abundant for the complex. In contrast, in the positive ion ESI mass spectra of apoCaM and Ca4CaM, the most abundant species in each case was the 8+ ion. When a complex of Ca4CaMwith TFP was prepared, the most abundant species was the 5+ ion. This is consistent with a conformational change of Ca4CaM that rendered some basic sites inaccessible to ionization in the ESI process. Using the same Ca4CaM/TFP mixture, no complex with TFP was observed in negative ion ESI mass spectra. These observations are discussed in the context of the structural changes that are known to occur in calmodulin, and suggestions are made to explain the apparently conflicting data. The results reported here reflect on the validity of using differences in charge-state distributions observed in ESI mass spectra to assess conformational changes in proteins.     

Ca2+诱导的淀粉液化芽孢杆菌α-淀粉酶分子的生物活性和结构变化

在研究Ca²⁺对淀粉液化芽孢杆菌α-淀粉酶分子生物活性影响的基础上, 采用荧光光谱法和傅里叶变换红外光谱法研究了Ca²⁺诱导的酶分子结构变化. 结果表明, 当溶液中Ca²⁺浓度低于25.0 mmol/L时, Ca²⁺对酶分子具有激活作用; 而当Ca²⁺浓度高于25.0 mmol/L时, Ca²⁺对酶分子的生物活性具有抑制作用. 在Ca²⁺诱导的淀粉液化芽孢杆菌α-淀粉酶分子结构变化过程中, 酶分子仅发生二级结构的变化, 并不涉及其三级结构. 当Ca²⁺对酶分子具有激活作用时, 酶分子中的无规卷曲结构及β-折叠结构的含量下降, 而α-螺旋结构及β-转角结构的含量上升; 而当Ca²⁺对酶分子生物活性具有抑制作用时, 酶分子中的α-螺旋结构及β-转角结构的含量下降, 而无规卷曲结构及β-折叠结构的含量上升.     

Coumarin Modified Rhodamine Derivative： Fluorescent Chemosensor Selectively Recognizing Al^3＋ and Ca^2＋

A fluorescent probe with a coumarin moiety bound to rhodamine 6G hydrazide（l） was synthesized. Its sensing behavior toward various metal ions was investigated with fluorescence methods. Compound I displays different fluorescence emission responses to Al^3＋ and Ca^2＋ at the same excitation wavelength in the visible light region, while no changes occur after the addition of other metal ions. The binding ratios of the complexs of 1-Al^3＋ and 1-Ca^2＋ are both 2：1 according to the Job plot and high resolution mass spectrometer（HRMS） experiments. Moreover, emission spectrum of 1-Ca^2＋ complex and absorption spectrum of the rbodamine dyes overlap largely. When Al^3＋ was added to the 1-Ca2＋ system, calcium in complex 1-Ca2＋ can be displaced by Al^3＋, resulting in the output of another ratiometric sensing signal, which demonstrates that the 1-Ca^2＋ complex can be served as a new and effective fluorescence resonance energy transfer（FRET） donor for rhodamine derivatives.     

吸收光谱电化学测定铜的研究

West等人^[1-2]首先研究了镉、钴、铬、铜、铁、锰、镍、铅等金属离子和某些有机化合物在铂电极上的吸收光谱电化学特性,并导出定量分析公式,但迄今为止,此法还未用于实际样品分析。本文在以前工作基础上,用原子吸收分光光度计,着重试验了钠、钙、镁、铝等对铜的影响,直接测定了腐蚀剂和氯化钠中的铜,获得了良好的结果。     

分子动力学模拟电解质对阴非离子表面活性剂界面行为的影响

通过分子动力学(MD)方法研究了不同类型电解质对阴非离子表面活性剂C₁₂EO₃C油水界面性能的影响。运用z轴质量密度分布、径向分布函数、分子间相互作用配位数、空间分布函数及均方根位移五种模拟参数来分析电解质与阴非离子表面活性剂的相互作用情况。研究表明,三种离子的加入均对水分子与表面活性剂亲水基形成的水化层结构产生影响,且从微观层面验证三种离子对表面活性剂亲水基相互作用强度大小顺序为Na⁺ < Ca²⁺ < Mg²⁺。通过扩散模拟结果可以较好地解释离子加入对界面张力平衡时间的影响情况。这对指导实验方向、制订最佳复配方案具有重要意义。     

A highly selective chemosensor for copper ion based on ICT fluorescence

Simple structural compounds 1 to 3 were synthesized.The presence of Cu²⁺ resulted in the fluorescence and absorption spectra change of 1 and 2,which indicated that 1 and 2 showed a highly selective response to Cu²⁺ over other metal ions.However,3 showed no selectivity for metal ions,which means that the compound could bind with several metal ions,such as,Ni²⁺,Zn²⁺,Cd²⁺.Hg²⁺, Pb²⁺,Fe³⁺,Mg²⁺,Ca²⁺,and Co²⁺,except Cu²⁺ and Ag⁺.The different spectral responses were attributed to the difference in binding sites for 1 and 3.     

Spectrofluorometry of Ions and Small Molecules Based on Conformational Changes of Specific Oligonucleotides with o-Phthalaldehyde-β-mercaptoethanol

Specific oligonucleotides such as telomere DNA and aptamer often undergo conformational changes upon ligand binding. Composite reagent composed of o-phthalaldehyde and β-mercaptoethanol(OPAME) has been extensively applied to fluorescent detection of amino compounds based on the reaction of primary amino-group, herein we proposed a general spectrofluorometry for ions and small molecules due to conformational changes upon ligand binding taking K+ and ATP as examples. In a borate controlled buffer medium, telomere DNA could react with OPAME, giving a thio-subtituted isoindole compound with strong fluorescence emission at 455 nm when excited at 340 nm. It was found that however, the fluorescence emission was greatly reduced in the presence of K+ since the formation of the quadruplex structure inhibits the reaction activity of amino-groups of telomere DNA. In order to testify the general application of OPAME reagent based on the conformational change of oligonucleotides, we further proposed a sensitive method of ATP based on its highly selective interaction with ATP-aptamer. The above mentioned applications show that the spectrofluorometry with the aid of OPAME reagent is simple, label free that is expected to be potentially general for DNA conformational change-based target detection.     

Supported lipid bilayer formation by the giant vesicle fusion induced by vesicle–surface electrostatic attractive interaction

The effect of the electrostatic attractive force between giant unilamellar vesicles (GUVs) and the SiO₂ surface on the formation of a Ca²⁺-free supported lipid bilayer (SLB) was investigated by atomic force microscopy and fluorescence microscopy. When negatively charged GUVs were incubated for 1 h without Ca²⁺, the surface coverage of lipid bilayer was <1% on the SiO₂ surface. In contrast, a high coverage was obtained without addition of Ca²⁺ on the positively charged surface modified by aminopropyldimethylethoxysilane, and the coverage of SLBs decreased with increasing KCl concentrations. The thickness of the water layer under SLB was reduced by modification of APS.     

Raman spectroscopic study of the conformational change of 12-crown-4 due to cation capture

Raman spectra of the Li⁺, Na⁺, K⁺, NH⁺₄, Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Pb²⁺ complexes of 12-crown-4 and also 12-crown-4 in various states are observed. The spectra of 12-crown-4 change remarkably by complex formation with cations. Normal vibration calculations of various conformations of 12-crown-4 are carried out. On the basis of the observed spectra and the results of the calculations, the conformation of 12-crown-4 in the Li⁺, Na⁺, K⁺, NH⁺₄, Mg²⁺ complexes is found to have approximate D_2d symmetry, while that in the Ca²⁺, Sr²⁺, Ba²⁺ complexes is found to have approximate C_2V symmetry.     

Infrared spectroscopic study of solid films of poly(ethylene glycol) doped with cations

The infrared spectra of solid films, prepared on a silicon plate of poly(ethylene glycol) doped with various cations (Li⁺, Na⁺, K⁺, Ca²⁺ and Ba²⁺), have been measured. The spectral analysis indicates that the films are non-crystalline and essentially amorphous. The polymer chain is significantly disordered by assuming diverse conformational states. The interaction between the ether oxygens of the polymer and the cation, as estimated from the C---O stretching wavenumber, is correlated to the size of the cation; the interaction is stronger for the cation with larger size.     

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.     

花粉多肽对钙调素的拮抗作用研究

研究了在Ca²⁺存在下荞麦花粉肽及其类似物和丹磺酰标记的钙调素(D-CaM)的相互作用,结果表明,除肽BP-1外,都能与D-CaM结合而形成复合物。利用荧光光谱法测定了这些复合物的解离常数K_d.在所研究的多肽中以BP-13拮抗CaM作用最强,其K_d值为4.6×10^-2μmol/L,抑制钙依赖性磷酸二酯酶活性的IC₅₀为2.2μmol/L.我们还发现,当D-丙氨酸残基取代没有亲和性的BP-1中甘氨酸残基时,其亲和性明显提高。     

Tb3+ as Fluorescent Probe in the Study of the Interaction of Other Lanthanide Ions with Trichosanthin

The interaction of lanthanide (Ln³⁺)ions with trchosanthin(TCS)was investi-gated using Tb³⁺ as fluorescent probe.The metal-binding sites of the protein were probed by means of adding the Tb³⁺ into the protein solutions.If other Ln³⁺ ions exist in the Tb³⁺ protein complex system, they would compete against Tb³⁺ in the binding sites of the protein, and so the fluorescence intensity of Tb³⁺ decreased,which is called quenching effect.The quencliing effect is related to the ionic radii of the Ln³⁺ ions and energy transfer from Tb³⁺ protein to Ln³⁺ ions.Based on the ex-perimental results,a schematic of the energy transfer from protein-Tb³⁺ complex to protein-Ln³⁺ complex is suggested.