F1-ATPase Chemical-Mechanical Coupling Mechanisms

F1-ATP酶化学机械耦合机制

• 批准号: 8005216
• 负责人: WAYNE D FRASCH
• 金额: $ 18.02万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-31 至 2010-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8005216
• 关键词: ADP-AlF4; ATP Hydrolysis; ATP Synthesis Pathway; ATP phosphohydrolase; Affect; Affinity; Bacteria; Binding; Biological Assay; C10; Catalytic Domain; Cell membrane; Charge; Chemicals; Chloroplasts; Communication; Complex; Computer software; Coupling; Custom; Enzymes; Escherichia coli; Event; F1-ATPase; Fluorescence; Generations; Goals; Human; Hydrogen Bonding; Ions; Laboratories; Location; Measurement; Measures; Mechanics; Membrane; Metals; Microscope; Mitochondria; Modeling; Molecular Motors; Motion; Movement; Mutation; Noise; Nucleotides; Protons; Reaction; Research Personnel; Rotation; Signal Transduction; Sodium Chloride; Structure; Testing; Thermodynamics; Time; Torque; Tyrosine; Work; Writing; clinically relevant; driving force; innovation; mutant; programs; single molecule

The F1Fo ATP synthase is a multisubunit, membrane-bound enzyme that catalyzes the synthesis of the majority of cellular ATP.Mutations in several subunits in the human mitochondrial enzyme are of important clinical relevance. The enzyme works as a molecular motor that drives the rotary motion of some of the subunits. The enzymes from mitochondria, chloroplasts and the plasma membranes of bacteria are closely similar. In E. coli, F1 is composed of an (ap)3 ring that surrounds the y subunit, and also contains 5 and e subunits. The membrane embedded Fo is composed of a c10 subunit ring to which is attached the a subunit and two b subunits. During ATP synthesis, the movement of protons through Fo drives the rotation of the c10 subunit ring to which the y and esubunits are attached that forces sequential conformational changes in the (cc|3)3 ring and results in the synthesis of ATP from each of the three (ap)3 heterodimers. The hydrolysis of ATP can also drive the rotation of the y subunit in the opposite direction. When F1 molecules are attached to a microscope cover slip, and a probe that can be observed under a microscope is attached to the y subunit, single molecules can be observed to rotate upon addition of ATP. Our long term objective is to elucidate the mechanism of ATPase-driven rotation of the y subunit. We propose the induction mechanism as a working hypothesis for rotation that will be critically examined. This hypothesis posits that Coulombic potential originating from residues that form hydrogen bonds and salt bridges between the (a(3)3 ring and the y subunit contribute to the generation of y subunit torque. We will test this hypothesis by accomplishing the following aims. (1) The contribution of p Catch Loop-y Subunit interactions to y subunit rotation will be examined by measuring the effects of mutations that eliminate these interactions on the rate of y subunit rotation measured using an innovative single molecule assay that we developed for this purpose. (2) The contribution of other known (a(J)3 ring-y subunit interactions to y subunit rotation will be determined that include contacts at the y subunit N and C termini.(3) The contribution to rotation of (ap)3 ring residues that do not interact directly with the y subunit will be examined including those that communicate between the y subunit and the catalytic sites. (4) The contribution to y subunit rotation of (ap)3 ring-y subunit interactions that have not been identified in known crystal structures of F1 but are identified by tracking the rotational path of charged and polar y subunit residues around the inside of the (ap)3 ring.

F1Fo ATP 合酶是一种多亚基膜结合酶，可催化大多数物质的合成 细胞 ATP。人类线粒体酶的几个亚基的突变具有重要的临床意义。这 酶作为分子马达驱动一些亚基的旋转运动。酶来自 细菌的线粒体、叶绿体和质膜非常相似。在大肠杆菌中，F1 由 (ap)3 环包围 y 亚基，还包含 5 和 e 亚基。膜嵌入Fo的组成 一个 c10 亚基环，其上连接有 a 亚基和两个 b 亚基。在 ATP 合成过程中， 质子通过 Fo 驱动 c10 亚基环的旋转，y 和 e 亚基附着在 c10 亚基环上，从而迫使 (cc|3)3 环中的连续构象变化，导致从三个 (ap)3 中的每一个合成 ATP 异二聚体。 ATP的水解还可以驱动y亚基沿相反方向旋转。当F1 将分子附着在显微镜盖玻片上，并附着可在显微镜下观察的探针 y 亚基，可以观察到单个分子在添加 ATP 后旋转。我们的长期目标是阐明 ATP酶驱动y亚基旋转的机制。我们建议将诱导机制作为一种工作机制 将严格检验旋转的假设。该假设假设库仑势源自 (a(3)3 环和 y 亚基之间形成氢键和盐桥的残基有助于 y 子单元扭矩的产生。我们将通过实现以下目标来检验这一假设。 (1) p的贡献 Catch Loop-y 亚基与 y 亚基旋转的相互作用将通过测量突变的影响来检查 使用创新的单分子测定法消除这些对 y 亚基旋转速率的相互作用 我们为此目的而开发。 (2)其他已知的(a(J)3环-y亚基相互作用对y亚基旋转的贡献 将确定包括 y 亚基 N 和 C 末端的接触。(3) (ap)3 环旋转的贡献 将检查不与 y 亚基直接相互作用的残基，包括那些在 y 亚基之间通信的残基 亚基和催化位点。 (4) (ap)3环-y亚基相互作用对y亚基旋转的贡献 已在已知的 F1 晶体结构中被识别，但通过跟踪带电和极性 y 的旋转路径来识别 (ap)3 环内部的亚基残基。

项目成果

Single-molecule detection of DNA via sequence-specific links between F1-ATPase motors and gold nanorod sensors.

• DOI: 10.1039/b716744j
• 发表时间: 2008-02
• 期刊: Lab on a chip
• 影响因子: 6.1
• 作者: J. York;D. Spetzler;Fusheng Xiong;W. Frasch

Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases.

单旋转 F-ATP 合成酶的结构不对称性和动力学跛行。

• DOI: 10.3390/molecules24030504
• 发表时间: 2019
• 期刊: Molecules (Basel, Switzerland)
• 作者: Sielaff,Hendrik;Yanagisawa,Seiga;Frasch,WayneD;Junge,Wolfgang;Börsch,Michael
• 通讯作者: Börsch,Michael

Interactions between beta D372 and gamma subunit N-terminus residues gamma K9 and gamma S12 are important to catalytic activity catalyzed by Escherichia coli F1F0-ATP synthase.

β D372 和 γ 亚基 N 末端残基 γ K9 和 γ S12 之间的相互作用对于大肠杆菌 F1F0-ATP 合酶的催化活性很重要。

• DOI: 10.1021/bi047293j
• 发表时间: 2005
• 期刊: Biochemistry.
• 作者: Lowry,DavidS;Frasch,WayneD
• 通讯作者: Frasch,WayneD

Interactions among gamma R268, gamma Q269, and the beta subunit catch loop of Escherichia coli F1-ATPase are important for catalytic activity.

γ R268、γ Q269 和大肠杆菌 F1-ATP 酶的 β 亚基捕获环之间的相互作用对于催化活性非常重要。

• DOI: 10.1074/jbc.m309948200
• 发表时间: 2003
• 期刊: The Journal of biological chemistry
• 作者: Greene,MatthewD;Frasch,WayneD
• 通讯作者: Frasch,WayneD

Abundance of Escherichia coli F1-ATPase molecules observed to rotate via single-molecule microscopy with gold nanorod probes.

通过使用金纳米棒探针的单分子显微镜观察到大量的大肠杆菌 F1-ATPase 分子旋转。

• DOI: 10.1007/s10863-007-9114-x
• 发表时间: 2007
• 期刊: Journal of bioenergetics and biomembranes
• 影响因子: 3
• 作者: York,Justin;Spetzler,David;Hornung,Tassilo;Ishmukhametov,Robert;Martin,James;Frasch,WayneD
• 通讯作者: Frasch,WayneD