F1F0-ATP酶的超分子结构和功能的研究

通过X衍射晶体学法、荧光标记显微镜法、负染色体电子显微镜与单克隆抗体技术等对F1F0 ATP酶结构和功能的研究发现,在大肠杆菌、叶绿体和牛心线粒体中,F1F0 ATP酶复合体分别由8、9和16种不同的亚基组成.所有F1F0 ATP酶都有类似的结构,球状的F1和F0是由一个中心转轴和一个外围柄连接在一起.其中,中心转轴由γ和ε亚基组成,外围柄由b2(Ⅰ,Ⅱ)和δ亚基组成.在酶的催化过程中,α3β3亚基通过γ亚基与膜上的c亚基环相互作用,驱动ATP合成酶的中心转轴旋转.F1F0 ATP酶利用电化学质子梯度的能量,催化ADP(腺苷二磷酸)和Pi(无机磷酸盐)形成ATP(腺苷三磷酸).     

ATP合酶的研究进展

ATP合酶又称F0F1-ATP酶,是一个多亚基复合体,广泛存在于生物界,包括细菌的质膜、线粒体内膜和类囊体膜,可以催化能源物质ATP的合成。这种酶在生物能学、生物化学、物理学和纳米学领域受到重要关注。ATP合酶主要由两部分组成:一是水溶性的蛋白复合体F1,另外一个是疏水部分F0,两者都是转动马达。其中F1亚基由核基因编码,F0亚基由线粒体ATP6和ATP8基因以及核基因共同编码。该酶利用线粒体内膜两侧质子梯度差形成的势能,通过结合改变机理旋转合成ATP,也可逆水解ATP形成梯度差,达到了很高的能量转化效率。研究其对于能量代谢具有重要价值,国内外学者一直在对其进行探索。综合国内外文献对ATP合酶的功能、结构、研究历史和影响因素进行了阐述。     

F1-ATPase转动马达的随机动力学研究

通过建立物理模型研究了马达F1-ATPase转动的过程,得到理想条件下马达F1-ATPase转动的角速度随ATP浓度、摩擦系数的变化规律.同时,在热涨落环境下,计算了马达F1-ATPase向前和向后转动的概率之比,修正了理想条件下马达的转动模型,得到与实验吻合的结果.     

人工质子动力,K~ 、Ca~(2 )梯度和E.coli对谷氨酸的吸收

Mitchell的化学渗透学说认为,细菌的质膜对于离子,特别是H~ 和OH~-离子,是不可透的。但是,质膜内的能量转换系统,如电子传递链和ATP酶复合体则有质子泵的功能,就是说,在底物氧化或ATP水解时,这些系统能从细胞内泵出质子,结果在质膜内外两侧建立起pH梯度和膜电位,二者之和构成了质子动力(Proton motiveforce)。当质膜外的质子重返细胞内时,这种质子动力可以推动膜内的耗能过程:借助ATP酶复合体来合成ATP,或借助运输蛋白系统来推动溶质的吸收。     

植物的光合作用(续三)——Ⅳ.ATP合成与电子传递的能量共轭机构

叶绿素吸收的光能,通过作用中心,转化为电能和还原力,从而使NADP~+还原。与此同时,在位于膜上的ATP合成酶(CF_0F_1)作用下形成ATP(图1)。ATP合成酶是从电子传递中获得能量的。1966年Mitchell首先用化学渗透学说来解释ATP合成的机理,即能量的传递和转化是通过H~+横切膜的运动来实现的。这一新的理论,对于促进人们对能量传递共轭机构的研究起到了有力的推动作用,促使科学家们重新对能量转化的共轭机构、膜的结构、离子运输等一系列问题在新理论的指导下开展研究。化学渗透学说     

凸极式永磁同步电机电磁功率和电磁转矩

永磁电机是联系机械系统和电系统的一种旋转机械。而机械系统和电系统是通过磁场的作用联系在一起。也就是说磁场是机械系统和电系统两者转换的媒介;是电磁功率(电磁转矩)产生的根源。电机学中以双反应理论为基础,求出凸极同步电机的电磁转矩公式。但是它把三相绕组的自感、互感关系一并考虑在合成磁势之中,掩盖了电机各感应系数随时间周期变化的物理实质,这对进一步研究电机在过渡过程及其复杂运行下电磁转矩、能量关系等均有局限性。因此我们从磁场能量相绕组之间的耦合关系,推导出凸极同步电机电磁转矩公式,有助于我们理解同步电机内部的电磁过程。 永磁同步电机的转子磁场是永久磁铁产生的,该转子磁场对定子线圈和铁芯的影响是与电磁铁等效的,我们便可以用一个近似的等效方法计算转子为永久磁铁的电机,用磁场能量和绕组之间的耦合关系,推导出凸极式永磁同步电机电磁转矩公式。     

利用酵母双杂交系统筛选油菜ATP6的相互作用因子

甘蓝型油菜波利马(Brassica napus pol.)细胞质雄性不育系是一种很有实用价值的油菜不育类型.目前关于其分子机制的研究已证实,线粒体ATP合成酶的一个亚基ATP6与其雄性不育相关.本实验以ATP6作为诱饵蛋白,应用酵母双杂交共转化的方法筛选与ATP6相互作用的蛋白质.在获得的阳性克隆中,发现了一个与拟南芥中未知基因AT3G47550所编码的蛋白质具有较高同源性的蛋白质.它含有RING HC的结构,可能在泛素-蛋白质降解途径与细胞质雄性不育间建立起了某种联系.     

小麦atpC基因植物超量表达载体的构建

线粒体ATP合成酶是氧化磷酸化过程中ATP合成起关键作用的多亚基复合体,但近来发现它们在植物应对非生物胁迫反应中起着十分重要的作用。因此,对ATP合成酶的各亚基基因功能的研究有助于阐释其在植物逆境条件下的调节机制,并为研究植物抗逆和防卫反应提供新的途径。线粒体ATP合成酶是能量代谢的关键酶,参与氧化磷酸化反应。atpC基因所编码的线粒体ATP合成酶γ亚基是线粒体ATP合成酶的功能亚基。为了进一步研究它在胁迫反应中的功能,以小麦cDNA为模板,通过RT-PCR方法扩增出atpC基因。将该基因的cDNA编码序列连接到pCAMBIA1301中,成功构建了小麦pCAMBIA-atpC植物超量表达载体,为后续的转基因研究奠定了基础。     

油菜细胞质雄性不育相关基因的筛选

甘蓝型油菜波利马(Brassica napus pol.)细胞质雄性不育系是一种很有实用价值的油菜不育类型.目前关于其分子机制的研究已证实,线粒体ATP合成酶的一个亚基ATP6与其雄性不育相关.本实验以ATP6作为诱饵蛋白,应用酵母双杂交共转化的方法筛选与ATP6相互作用的蛋白质.在获得的阳性克隆中,发现了一个与tAPX基因及其表达的蛋白都具有较高同源性.它是活性氧清除的重要酶类,很可能与细胞质雄性不育间建立起了某种联系.     

线粒体F型ATP合成酶的制备方法研究与探讨

科研工作者们在过去的50年前赴后继的工作中深入研究了ATP合成酶的功能,并努力尝试解析该酶的空间结构以便能够从结构基础上对ATP合成酶的催化机理进行阐明;但蛋白的纯化工作却一直是困扰研究顺利进展的最大障碍.蛋白的纯化技术是与特定阶段科技发展及科研工作者思维模式的直接反应,因而是一门不断发展的艺术.文章主要介绍了ATP合成酶的提取与纯化工作,在详细对比了现有方法的基础上,给出了纯化此酶复合体的详细方案和线粒体、亚线粒体、ATP合成酶的提取与纯化方法;并在方法讨论的基础上对线粒体F型ATP合成酶的研究提出了前瞻性的方案.     

Structural changes linked to proton translocation by subunit c of the ATP synthase

F1F0 ATP synthases use a transmembrane proton gradient to drive the synthesis of cellular ATP. The structure of the cytosolic F1 portion of the enzyme and the basic mechanism of ATP hydrolysis by F1 are now well established, but how proton translocation through the transmembrane F0 portion drives these catalytic changes is less clear. Here we describe the structural changes in the proton-translocating F0 subunit c that are induced by deprotonating the specific aspartic acid involved in proton transport. Conformational changes between the protonated and deprotonated forms of subunit c provide the structural basis for an explicit mechanism to explain coupling of proton translocation by F0 to the rotation of subunits within the core of F1. Rotation of these subunits within F1 causes the catalytic conformational changes in the active sites of F1 that result in ATP synthesis.     

E. coli F1-ATPase interacts with a membrane protein component of a proton channel

The ATP synthases of bacteria, mitochondria and chloroplasts, which use the energy of a transmembrane proton gradient to power the synthesis of ATP, consist of an integral membrane component F0--thought to contain a proton channel--and a catalytic component, F1. To help investigate the way F0 and F1 are coupled, we have sequenced the b-subunit of the Escherichia coli F0, which seems to be the counterpart of a thermophilic bacteria F0 subunit thought to be essential for F1 binding. We report here that its sequence is remarkable, being hydrophobic around the N-terminus and highly charged in the remainder. We propose that the N-terminal segment lies in the membrane and the rest outside. The extramembranous section contains two adjacent stretches of 31 amino acids where the sequence is very similar: in the second of these stretches there is further internal homology. These duplicated stretches of the polypeptide probably fold into two alpha-helices which have many common features able to make contact with F1 subunits. Thus protein b occupies a central position in the enzyme, where it may be involved in proton translocation. It is possibly also important in biosynthetic assembly.     

Subnanometre-resolution structure of the intact Thermus thermophilus H+-driven ATP synthase

Ion-translocating rotary ATPases serve either as ATP synthases, using energy from a transmembrane ion motive force to create the cell's supply of ATP, or as transmembrane ion pumps that are powered by ATP hydrolysis. The members of this family of enzymes each contain two rotary motors: one that couples ion translocation to rotation and one that couples rotation to ATP synthesis or hydrolysis. During ATP synthesis, ion translocation through the membrane-bound region of the complex causes rotation of a central rotor that drives conformational changes and ATP synthesis in the catalytic region of the complex. There are no structural models available for the intact membrane region of any ion-translocating rotary ATPase. Here we present a 9.7?? resolution map of the H(+)-driven ATP synthase from Thermus thermophilus obtained by electron cryomicroscopy of single particles in ice. The 600-kilodalton complex has an overall subunit composition of A(3)B(3)CDE(2)FG(2)IL(12). The membrane-bound motor consists of a ring of L subunits and the carboxy-terminal region of subunit I, which are equivalent to the c and a subunits of most other rotary ATPases, respectively. The map shows that the ring contains 12 L subunits and that the I subunit has eight transmembrane helices. The L(12) ring and I subunit have a surprisingly small contact area in the middle of the membrane, with helices from the I subunit making contacts with two different L subunits. The transmembrane helices of subunit I form bundles that could serve as half-channels across the membrane, with the first half-channel conducting protons from the periplasm to the L(12) ring and the second half-channel conducting protons from the L(12) ring to the cytoplasm. This structure therefore suggests the mechanism by which a transmembrane proton motive force is converted to rotation in rotary ATPases.     

Mechanically driven ATP synthesis by F1-ATPase

ATP, the main biological energy currency, is synthesized from ADP and inorganic phosphate by ATP synthase in an energy-requiring reaction. The F1 portion of ATP synthase, also known as F1-ATPase, functions as a rotary molecular motor: in vitro its gamma-subunit rotates against the surrounding alpha3beta3 subunits, hydrolysing ATP in three separate catalytic sites on the beta-subunits. It is widely believed that reverse rotation of the gamma-subunit, driven by proton flow through the associated F(o) portion of ATP synthase, leads to ATP synthesis in biological systems. Here we present direct evidence for the chemical synthesis of ATP driven by mechanical energy. We attached a magnetic bead to the gamma-subunit of isolated F1 on a glass surface, and rotated the bead using electrical magnets. Rotation in the appropriate direction resulted in the appearance of ATP in the medium as detected by the luciferase-luciferin reaction. This shows that a vectorial force (torque) working at one particular point on a protein machine can influence a chemical reaction occurring in physically remote catalytic sites, driving the reaction far from equilibrium.     

Structure of the C-terminal domain of FliG, a component of the rotor in the bacterial flagellar motor.

Many motile species of bacteria are propelled by flagella, which are rigid helical filaments turned by rotary motors in the cell membrane. The motors are powered by the transmembrane gradient of protons or sodium ions. Although bacterial flagella contain many proteins, only three-MotA, MotB and FliG-participate closely in torque generation. MotA and MotB are ion-conducting membrane proteins that form the stator of the motor. FliG is a component of the rotor, present in about 25 copies per flagellum. It is composed of an amino-terminal domain that functions in flagellar assembly and a carboxy-terminal domain (FliG-C) that functions specifically in motor rotation. Here we report the crystal structure of FliG-C from the hyperthermophilic eubacterium Thermotoga maritima. Charged residues that are important for function, and which interact with the stator protein MotA, cluster along a prominent ridge on FliG-C. On the basis of the disposition of these residues, we present a hypothesis for the orientation of FliG-C domains in the flagellar motor, and propose a structural model for the part of the rotor that interacts with the stator.     

Direct observation of steps in rotation of the bacterial flagellar motor

The bacterial flagellar motor is a rotary molecular machine that rotates the helical filaments that propel many species of swimming bacteria. The rotor is a set of rings up to 45 nm in diameter in the cytoplasmic membrane; the stator contains about ten torque-generating units anchored to the cell wall at the perimeter of the rotor. The free-energy source for the motor is an inward-directed electrochemical gradient of ions across the cytoplasmic membrane, the protonmotive force or sodium-motive force for H+-driven and Na+-driven motors, respectively. Here we demonstrate a stepping motion of a Na+-driven chimaeric flagellar motor in Escherichia coli at low sodium-motive force and with controlled expression of a small number of torque-generating units. We observe 26 steps per revolution, which is consistent with the periodicity of the ring of FliG protein, the proposed site of torque generation on the rotor. Backwards steps despite the absence of the flagellar switching protein CheY indicate a small change in free energy per step, similar to that of a single ion transit.     

Highly coupled ATP synthesis by F1-ATPase single molecules

F1-ATPase is the smallest known rotary motor, and it rotates in an anticlockwise direction as it hydrolyses ATP. Single-molecule experiments point towards three catalytic events per turn, in agreement with the molecular structure of the complex. The physiological function of F1 is ATP synthesis. In the ubiquitous F0F1 complex, this energetically uphill reaction is driven by F0, the partner motor of F1, which forces the backward (clockwise) rotation of F1, leading to ATP synthesis. Here, we have devised an experiment combining single-molecule manipulation and microfabrication techniques to measure the yield of this mechanochemical transformation. Single F1 molecules were enclosed in femtolitre-sized hermetic chambers and rotated in a clockwise direction using magnetic tweezers. When the magnetic field was switched off, the F1 molecule underwent anticlockwise rotation at a speed proportional to the amount of synthesized ATP. At 10 Hz, the mechanochemical coupling efficiency was low for the alpha3beta3gamma subcomplex (F1-epsilon)), but reached up to 77% after reconstitution with the epsilon-subunit (F1+epsilon)). We provide here direct evidence that F1 is designed to tightly couple its catalytic reactions with the mechanical rotation. Our results suggest that the epsilon-subunit has an essential function during ATP synthesis.     

Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase

The enzyme F1-ATPase has been shown to be a rotary motor in which the central gamma-subunit rotates inside the cylinder made of alpha3beta3 subunits. At low ATP concentrations, the motor rotates in discrete 120 degrees steps, consistent with sequential ATP hydrolysis on the three beta-subunits. The mechanism of stepping is unknown. Here we show by high-speed imaging that the 120 degrees step consists of roughly 90 degrees and 30 degrees substeps, each taking only a fraction of a millisecond. ATP binding drives the 90 degrees substep, and the 30 degrees substep is probably driven by release of a hydrolysis product. The two substeps are separated by two reactions of about 1 ms, which together occupy most of the ATP hydrolysis cycle. This scheme probably applies to rotation at full speed ( approximately 130 revolutions per second at saturating ATP) down to occasional stepping at nanomolar ATP concentrations, and supports the binding-change model for ATP synthesis by reverse rotation of F1-ATPase.     

转子流道倾斜式的外驱旋转压能交换器性能研究

为提高外驱式旋转压能交换器性能，设计了转子流道倾斜式的外驱式旋转压能交换器，以能量回收效率和体积混合率为性能指标，利用商业CFD软件Fluent对模型求解计算，并与直流道外驱式旋转压能交换器模拟数据对比分析。结果表明，处理量相等的情况下，能量回收效率和体积混合率均随转子转速的升高而降低；同一转速下，转子倾斜角度改变，能量回收效率变化不超过0.04%，而对体积混合率有明显影响。研究结果对于提高外驱式旋转压能交换器设备性能具有一定指导意义。     

转子流道倾斜式的外驱旋转压能交换器性能研究

为提高外驱式旋转压能交换器性能,设计了转子流道倾斜式的外驱式旋转压能交换器,以能量回收效率和体积混合率为性能指标,利用商业CFD软件Fluent对模型求解计算,并与直流道外驱式旋转压能交换器模拟数据对比分析。结果表明,处理量相等的情况下,能量回收效率和体积混合率均随转子转速的升高而降低;同一转速下,转子倾斜角度改变,能量回收效率变化不超过0.04%,而对体积混合率有明显影响。研究结果对于提高外驱式旋转压能交换器设备性能具有一定指导意义。