The Peripheral Stalk of F1F0 ATP Synthase

描述(申请人提供)：我们实验室的长期目标是了解H+转运与F1F0 ATP合成酶催化活性之间的耦合机制。这种酶起着能量产生的中心代谢作用。F1F0ATP合成酶家族的一个成员存在于植物、动物和细菌中。F1F0ATP合成具有共同的分子结构和机制，因此对一个家族成员的观察通常适用于所有成员。在人类中，能量代谢缺陷与复杂的疾病有关，包括衰老、阿尔茨海默病、癌症和糖尿病。不同的症状被归因于FIF0-ATP合成酶的特定遗传缺陷。因此，确实需要了解突变对酶机制的影响。这项建议的主要目的是确定F1F0ATP合成酶的外周茎是否在F1F0ATP合成酶的机制中发挥动态作用。具体目的1研究外周茎中含有突变b亚基的纯化的F1F0 ATP合成酶的活性。这些实验将辅以遗传学研究，以确定能够相互补充的成对突变的uncF(B)基因。特定目标2着眼于改变的B亚基在外周茎内的定位。特定目标3确定催化活性是否改变外围茎的取向。这些实验包括构建包含嵌合b亚基的F1F0 ATP合成酶，以便在酶处于静止、被抑制或正在进行催化周转时确定两个b亚基的相对位置。具体目标4考虑了催化过程中秸秆的物理状态。荧光环境的探针将定位在外周茎中的一个b亚基上，并在酶静止或催化ATP水解时记录变化。FRET策略将被用来考虑包含不同长度的外围柄的FIFO复合体的结构变化。

DESCRIPTION (provided by applicant): The long term goal of our laboratory is to understand the mechanism coupling H+ translocation to catalytic activity in F1F0 ATP synthase. The enzyme serves the central metabolic role of energy production. A member of the F1F0 ATP synthase family is found in plants, animals and bacteria. The F1F0 ATP syntheses share a common molecular architecture and mechanism, so observations on one family member are generally applicable to all. In humans, defects in energy metabolism have been implicated in complex conditions including aging, Alzheimer's disease, cancer and diabetes. Distinct syndromes are attributed to specific inherited genetic defects in FIF0 ATP synthase. Therefore, a real need exists to understand the effects of mutations on the mechanism of the enzyme. The major goal of this proposal is to determine if the peripheral stalk of F1F0 ATP synthase plays a dynamic role in the mechanism of F1F0 ATP synthase. Specific Aim 1 studies the activity in purified F1F0 ATP synthases enzymes containing mutant b subunits in the peripheral stalk. These experiments will be supplemented with genetic studies to identify pairs of mutant uncF(b) genes capable of complementing each other. Specific Aim 2 looks at the positioning of the altered b subunits within the peripheral stalk. Specific Aim 3 determines if catalytic activity alters the orientation of the peripheral stalk. The experiments involve construction of F1F0 ATP synthase containing chimeric b subunits to allow determination of the relative positions of the two b subunits when the enzyme is at rest, inhibited or undergoing catalytic turnover. Specific Aim 4 considers the physical state of the stalk during catalysis. Probes of fluorescence environment will be positioned on one b subunit in the peripheral stalk and changes recorded while the enzyme is at rest or catalyzing ATP hydrolysis. A FRET strategy will be used to consider changes in the structure of FIFO complexes containing peripheral stalks of various lengths.