分子动力学研究F1-ATP合酶对三磷酸腺苷的稳定和定位作用

F₁-ATP合酶通过与ATP之间建立广泛的相互作用,实现对ATP的位置进行精确的定位.这些相互作用为ATP的合成/水解创造了稳定的环境.理解这些相互作用是理解ATP的合成/水解机理的基础.我们通过分子动力学模拟方法研究这些相互作用,找出在稳定化过程中起到重要作用的残基.通过检测ATP和F₁-ATP合酶之间的非键相互作用,发现残基段158-164所形成的loop区域及残基R189, Y345对ATP存在显著相互作用.其中,该loop区域对ATP的三磷酸部分形成一个半包围结构,封闭活性位点区域,并通过氢键网络约束ATP三磷酸的运动,为ATP合成/水解创造稳定的环境.此外,关键残基Y345通过π-π叠加相互作用对ATP的碱基进行约束,但是ATP的碱基可以在平行于Y345芳香环的平面内进行滑动,我们推断这种滑动运动有利于促进ATP的水解.     

A Multisite‐Binding Switchable Fluorescent Probe for Monitoring Mitochondrial ATP Level Fluctuation in Live Cells

Adenosine triphosphate (ATP), commonly produced in mitochondria, is required by almost all the living organisms; thus fluorescent probes for monitoring mitochondrial ATP levels fluctuation are essential and highly desired. Herein, we report a multisite‐binding switchable fluorescent probe, ATP‐Red 1 , which selectively and rapidly responds to intracellular concentrations of ATP. Live‐cell imaging indicated that ATP‐Red 1 mainly localized to mitochondria with good biocompatibility and membrane penetration. In particular, with the help of ATP‐Red 1 , we successfully observed not only the decreased mitochondrial ATP levels in the presence of KCN and starvation state, but also the increased mitochondrial ATP levels in the early stage of cell apoptosis. These results indicate that ATP‐Red 1 is a useful tool for investigating ATP‐relevant biological processes.     

Ratiometric fluorescence and visual sensing of ATP based on gold nanocluster-encapsulated metal-organic framework with a smartphone

Adenosine triphosphate (ATP) plays an important role in various biological processes and the ATP level is closely associated with many diseases. Herein, we designed a novel dual-emissive fluorescence nanoplatform for ATP sensing based on red emissive europium metal-organic framework (Eu-MOF) and blue emissive gold nanoclusters (AuNCs). The presence of ATP causes the decomposition of Eu-MOF owing to strong affinity of Eu³⁺ with ATP. As a result, the red emission of Eu-MOF decreases while the blue emission of AuNCs remains unchanged. The distinct red/blue emission intensity change enables the establishment of a ratiometric fluorescent and visual sensor of ATP. Moreover, a fluorescent paper-based sensor was fabricated with the ratiometric ATP probes, which enabled easy-to-use and visual detection of ATP in serum samples with a smartphone.     

Enhanced Anticancer Efficacy by ATP‐Mediated Liposomal Drug Delivery

A liposome‐based co‐delivery system composed of a fusogenic liposome encapsulating ATP‐responsive elements with chemotherapeutics and a liposome containing ATP was developed for ATP‐mediated drug release triggered by liposomal fusion. The fusogenic liposome had a protein–DNA complex core containing an ATP‐responsive DNA scaffold with doxorubicin (DOX) and could release DOX through a conformational change from the duplex to the aptamer/ATP complex in the presence of ATP. A cell‐penetrating peptide‐modified fusogenic liposomal membrane was coated on the core, which had an acid‐triggered fusogenic potential with the ATP‐loaded liposomes or endosomes/lysosomes. Directly delivering extrinsic liposomal ATP promoted the drug release from the fusogenic liposome in the acidic intracellular compartments upon a pH‐sensitive membrane fusion and anticancer efficacy was enhanced both in vitro and in vivo.     

A Novel Fluorescent Probe for ATP Detection Based on Synergetic Effect of Aggregation-induced Emission and Counterion Displacement

In this work,a fluorescent probe(TPEBe-I)was developed for adenosine triphosphate(ATP)detection based on the synergetic effect of aggregation-induced emission and counterion displacement.TPEBe-I gave weak emission in aqueous solution due to the heavy-atom effect of counter iodide ion.However,upon the addition of ATP,the new aggregate complex(TPEBe-ATP)was formed between the cationic unit of TPEBe-I and ATP through electrostatic interactions,which not only restricted the intramolecular motion of luminogen but also eliminated the quenching effect of iodide ion.As a result,the fluorescent light-up detection for ATP was successfully achieved.Moreover,TPEBe-I exhibited high selectivity towards ATP and showed a wide linear detection region towards the logarithm of ATP concentration(5—600μmol/L)with a detection limit of 1.0μmol/L,enabling TPEBe-I as a promising probe for ATP quantitative analysis.     

Water‐Soluble Triarylboron Compound for ATP Imaging In Vivo Using Analyte‐Induced Finite Aggregation

Adenosine 5′‐triphosphate (ATP) is a multifunctional molecule that participates in many important biological processes. Currently, fluorescence indicators for ATP with high performance are in demand. Reported herein is a novel water‐soluble triarylboron compound which displays an apparent ATP‐dependent fluorescence enhancement when dispersed in water. It can selectively recognize ATP from other bioactive substances in vitro and in vivo. The ATP‐induced finite aggregation endows the indicator with appreciable photostability and superior tolerance to environmental electrolytes. This indicator has been successfully applied to the ATP imaging in NIH/3T3 fibroblast cells. The difference in the ATP levels within the membrane and cytosol is clearly visible.     

Electrochemical/electrospray mass spectrometric studies of electrochemically stimulated ATP release from PP/ATP films

Polypyrrole (PP)/adenosine triphosphate (ATP) was chosen as a conducting polymer/anionic drug model to serve as a bioactive releasing material for ATP. The process of ATP release from PP/ATP films was investigated for the first time by electrochemical electrospray ionization mass spectrometry (EC/ESMS). This technique allowed the simultaneous and direct detection of ATP and its related species during electrochemical release. In the experiments, suitable solvent conditions were found for both the electrochemical release and the electrospray mechanisms. EC/ESMS results showed that continuous potential cycles allowed a higher ATP release rate than potential steps. It was also found that the film thickness is an important factor affecting the rate and the amount of electrochemical ATP release.     

Extracellular ATP is generated by ATP synthase complex in adipocyte lipid rafts

Mitochondrial biogenesis is known to accompany adipogenesis to complement ATP and acetyl-CoA required for lipogenesis. Here, we demonstrated that mitochondrial proteins such as ATP synthase alpha and beta, and cytochrome c were highly expressed during the 3T3-L1 differentiation into adipocytes. Fully-differentiated adipocytes showed a significant increase of mitochondria under electron microscopy. Analysis by immunofluorescence, cellular fractionation, and surface biotinylation demonstrated the elevated levels of ATP synthase complex found not only in the mitochondria but also on the cell surface (particularly lipid rafts) of adipocytes. High rate of ATP (more than 30 microM) synthesis from the added ADP and P(i) in the adipocyte media suggests the involvement of the surface ATP synthase complex for the extracellular ATP synthesis. In addition, this ATP synthesis was significantly inhibited in the presence of oligomycin, an ATP synthase inhibitor, and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an ATP synthase uncoupler. Decrease of extracellular ATP synthesis in acidic but not in basic media further indicates that the surface ATP synthase may also be regulated by proton gradient through the plasma membrane.     

Intramitochondrial co-assembly between ATP and nucleopeptides induces cancer cell apoptosis

Mitochondria are essential intracellular organelles involved in many cellular processes, especially adenosine triphosphate (ATP) production. Since cancer cells require high ATP levels for proliferation, ATP elimination can be a unique target for cancer growth inhibition. We describe a newly developed mitochondria-targeting nucleopeptide (MNP) that sequesters ATP by self-assembling with ATP inside mitochondria. MNP interacts strongly with ATP through electrostatic and hydrogen bonding interactions. MNP exhibits higher binding affinity for ATP (−637.5 kJ mol⁻¹) than for adenosine diphosphate (ADP) (−578.2 kJ mol⁻¹). To improve anticancer efficacy, the small-sized MNP/ADP complex formed large assemblies with ATP inside cancer cell mitochondria. ATP sequestration and formation of large assemblies of the MNP/ADP–ATP complex inside mitochondria caused physical stress by large structures and metabolic disorders in cancer cells, leading to apoptosis. This work illustrates a facile approach to developing cancer therapeutics that relies on molecular assemblies.

Mitochondria-targeting nucleopeptide (MNP) can sequester ATP by self-assembling with ATP. A small nanosized MNP/ADP complex forms a large assembly with ATP. Thus, intramitochondrial co-assembly causes stress by large structures and apoptosis.     

Determination of ATP using a double-receptor sandwich method based on molecularly imprinted membrane and fluorescence-labeled uranyl–salophen complex

A double-receptor sandwich method for the fluorescence determination of adenosine triphosphate (ATP) is proposed in this paper. The solid phase receptor on the surface of glass slides is a molecularly imprinted membrane (MIM) containing an artificial nanocavity. It is constructed by a molecular imprinting technique using adenosine monophosphate (AMP) as a template molecule. The labeled receptor is a uranyl–salophen complex containing a fluorescent group or uranyl–salophen–fluorescein (USF). It is synthesized with salophen, 5-aminofluorescein, and uranyl. In a procedure of determining ATP, ATP in sample solution is first adsorbed on the surface of the glass slide through the combination of the AMP group in ATP with the nanocavity in MIM. Then, the adsorbed ATP binds USF through the coordination reaction of the phosphate group in ATP with uranyl in USF to form a sandwich-type structure of MIM-ATP-USF. The amount of ATP is detected through the fluorescence determination of USF bound on the slide. Under optimal conditions, the linear range for the determination of ATP is 0.3 to 4.8 nmol/mL with a detection limit of 0.041 nmol/mL. The proposed method has been successfully employed for the determination of ATP in real samples with the recoveries of 98.5 to 102.5 %.     

Isolation of ATP from a yeast fermentation broth using a cryogel column at high flow velocities

This communication presents an effective method for isolating adenosine triphosphate (ATP) from a yeast fermentation broth using an anion‐exchange supermacroporous cryogel column at high flow velocities. The breakthrough and elution behaviors of pure ATP in the cryogel bed were investigated at flow velocities of 2, 5, and 10 cm/min and the ATP binding capacities were determined. Then the ATP‐containing yeast fermentation broth was employed as the test feedstock and various chromatographic runs were conducted to isolate ATP by the cryogel at different high flow velocities. The ATP samples obtained were analyzed quantitatively by HPLC. The results showed that even at a flow velocity of 5 or 10 cm/min, a product purity of 97.4 or 98.0% can be achieved, illustrating the potential of the present method for separation of high‐purity ATP directly from fermentation feedstock at high flow velocities.     

Irreversibly electropermeabilized yeast retains the capability for ATP synthesis via oxidative phosphorylation

ATP synthesis in irreversibly electropermeabilized yeast Kluyveromyces lactis was studied by using different respiratory substrates. The permeabilization itself provoked a dramatic decrease of the total ATP level and the cells lost their ability to synthesize ATP via glycolysis. The addition of exogenous NADH supported ATP synthesis in irreversibly permeabilized cells for up to 4-6 h after substrate addition when the total ATP level became twice that of intact cells incubated for the same period with lactose.     

Measuring the simultaneous effects of hypoxia and deformation on ATP release from erythrocytes

It is known that adenosine triphosphate (ATP) is released from red blood cells (RBCs) due to various forms of stimulation such as deformation, pharmacological stimuli, and hypoxia. To date, these various stimuli have been investigated individually. Here, we have combined a microflow system capable of initiating deformation-induced release of ATP from the RBCs at various levels of hypoxia as measured by percent oxygen saturation in the RBC sample. When values of ATP released from deformation and hypoxia are compared to values of ATP release due to hypoxia alone, the relationship between the two stimuli can be deduced. Measurement of RBC-derived ATP with the well-known chemiluminescence assay employing luciferin/luciferase indicates that RBCs deoxygenated for 4 min released 1.84 +/- 0.075 microM ATP. The largest decrease in oxygen saturation was found to be between 0 s (66.3% O(2) saturation) and 15 s (22.3% O(2) saturation). RBCs deoxygenated to a 22.3% O(2) saturation released 0.374 +/- 0.011 microM ATP when pumped through the microflow system. This value is an increase from 0.281 +/- 0.007 microM ATP in the presence of flow alone. The ATP release after exposure to hypoxia at 22.3% O(2) saturation was 0.381 +/- 0.014 microM ATP, a value statistically equivalent to that of hypoxia and flow combined. These data suggest that, at an oxygen saturation point of around 25.0% or above, deformation contributes to ATP release from the RBC; however, beyond this saturation point, the ATP release is largely due to hypoxia.     

Role of the D-loops in allosteric control of ATP hydrolysis in an ABC transporter

ABC transporters couple ATP hydrolysis to movement of substrates across cell membranes. They comprise two transmembrane domains and two cytosolic nucleotide-binding domains forming two active sites that hydrolyze ATP cooperatively. The mechanism of ATP hydrolysis is controversial and the structural dynamic basis of its allosteric control unknown. Here we report molecular dynamics simulations of the ATP/apo and ATP/ADP states of the bacterial ABC exporter Sav1866, in which the cytoplasmic region of the protein was simulated in explicit water for 150 ns. In the simulation of the ATP/apo state, we observed, for the first time, conformers of the active site with the canonical geometry for an in-line nucleophilic attack on the ATP γ-phosphate. The conserved glutamate immediately downstream of the Walker B motif is the catalytic base, forming a dyad with the H-loop histidine, whereas the Q-loop glutamine has an organizing role. Each D-loop provides a coordinating residue of the attacking water, and comparison with the simulation of the ATP/ADP state suggests that via their flexibility, the D-loops modulate formation of the hydrolysis-competent state. A global switch involving a coupling helix delineates the signal transmission route by which allosteric control of ATP hydrolysis in ABC transporters is mediated.     

聚丙烯/凹凸棒石纳米复合材料的制备与性能研究

以聚丙烯(PP)为聚合物基体,天然凹凸棒石(ATP)为无机组分,经过氧化聚乙烯对ATP表面进行包覆处理,用熔融共混的方法制备了PP/ATP纳米复合材料.扫描电镜结果显示,经本方法处理后的ATP在PP基体中分散较为均匀.ATP棒晶簇直径最佳分散尺寸能达到20~40 nm,比未处理ATP在基体中的棒晶簇直径小10 nm以上;XRD测试表明,未处理ATP和处理后的ATP均有使PP晶粒细化的作用,同时不改变PP的α晶型;DSC结果显示,ATP的加入提高了PP的结晶温度和结晶度,说明ATP有一定的成核作用.通过对复合材料的力学性能测试发现,经过处理的ATP制备的复合材料力学性能优于未处理ATP复合材料对PP力学性能的改善.其中ATP与氧化聚乙烯固含量的质量比为2∶1,ATP含量为3 wt%时复合材料力学性能达到最好.缺口冲击强度比纯PP最高提高了83%,提高幅度显著;经过处理的ATP制备的复合材料拉伸强度提高了6%~11%;弯曲强度提高了33%~45%;弯曲模量提高了90%~106%.     

基于微悬臂梁生物传感器检测三磷酸腺苷

基于适配子构建了无标记检测三磷酸腺苷(ATP)的微悬臂梁生物传感器。 将ATP适配子修饰在微悬臂梁阵列中的传感悬臂镀金面上,用来识别ATP,而参比悬臂修饰巯基己醇(MCH)防止非特异性吸附。 ATP与其适配子发生特异性相互作用,使悬臂的上下两个表面产生应力差,导致传感悬臂产生偏转,扣除参比悬臂偏转后其偏转值与ATP的浓度在0.5~5 mmol/L范围内有良好的线性关系,相关系数为0.998,最低检出限为0.06 mmol/L。 该微悬臂梁生物传感器响应快速、操作简单,并且对ATP具有良好的特异性。     

Enzyme-based field-effect transistor for adenosine triphosphate (ATP) sensing.

Adenosine triphosphate (ATP) not only functions as an energy-carrier substance and an informative molecule, but also acts as a marker substance in studies of both bio-traces and cellular/tissular viability. Due to the importance of the ATP function for living organisms, in situ assays of ATP are in demand in various fields, e.g., hygiene. In the present study, we developed an ATP sensor that combines the selective catalytic activity of enzyme and the properties of an ion selective field effect transistor (ISFET). In this system, the ATP hydrolyrase, "apyrase (EC 3.6.1.5.)" is encased in a gel and mounted on a Ta(2)O(5) ISFET gate surface. When the enzyme layer selectively catalyzes the dephosphorylation of ATP, protons are accumulated at the gate because the enzymatic reaction produces H(+) as a byproduct. Based on the interfacial enzymatic reaction, the response from the ISFET is completely dependent upon the ATP concentration in the bulk solution. This device is readily applicable to practical in situ ATP measurement, e.g. hygienic usage.     

Dynamic Control of Microbial Movement by Photoswitchable ATP Antagonists

Adenosine triphosphate (ATP) is the energy source for various biochemical processes and biomolecular motors in living things. Development of ATP antagonists and their stimuli-controlled actions offer a novel approach to regulate biological processes. Herein, we developed azobenzene-based photoswitchable ATP antagonists for controlling the activity of motor proteins; cytoplasmic and axonemal dyneins. The new ATP antagonists showed reversible photoswitching of cytoplasmic dynein activity in an in vitro dynein-microtubule system due to the trans and cis photoisomerization of their azobenzene segment. Importantly, our ATP antagonists reversibly regulated the axonemal dynein motor activity for the force generation in a demembranated model of Chlamydomonas reinhardtii. We found that the trans and cis isomers of ATP antagonists significantly differ in their affinity to the ATP binding site.     

A cholic acid-based fluorescent chemosenor for the detection of ATP

A novel ditopic cholic acid-based fluorescent chemosensor for ATP, 1a, was designed and synthesized. Its interactions with phosphates, AMP, ADP, ATP, CTP, GTP, and TTP have been investigated. When ATP was added to a 1:1 aqueous CH3CN solution of the sensor at pH 7.4, a significant decrease in fluorescence of 1a was observed, whereas other guest molecules showed a much smaller effect. The complex between 1a and ATP was confirmed through combined UV, 1H, 13C and 31P NMR spectroscopic methods. The uniqueness of the new sensor is that it binds with ATP 33-124 times more selectively than other nucleotides, as evidenced from the respective binding constants. 1a is a highly sensitive sensing probe; as little as 30 nM ATP can cause 15% fluorescence quenching of the sensor.     

ATP‐Mediated Transient Behavior of Stomatocyte Nanosystems

In nature, dynamic processes are ubiquitous and often characterized by adaptive, transient behavior. Herein, we present the development of a transient bowl‐shaped nanoreactor system, or stomatocyte, the properties of which are mediated by molecular interactions. In a stepwise fashion, we couple motility to a dynamic process, which is maintained by transient events; namely, binding and unbinding of adenosine triphosphate (ATP). The surface of the nanosystem is decorated with polylysine (PLL), and regulation is achieved by addition of ATP. The dynamic interaction between PLL and ATP leads to an increase in the hydrophobicity of the PLL–ATP complex and subsequently to a collapse of the polymer; this causes a narrowing of the opening of the stomatocytes. The presence of the apyrase, which hydrolyzes ATP, leads to a decrease of the ATP concentration, decomplexation of PLL, and reopening of the stomatocyte. The competition between ATP input and consumption gives rise to a transient state that is controlled by the out‐of‐equilibrium process.