Subunit and Nucleotide Interactions in the Chloroplast ATP Synthase

由腺苷二磷酸（ADP）和无机磷酸（Pi）合成腺苷三磷酸（ATP）是由叶绿体、线粒体和某些细菌的能量传递膜中密切相关的酶催化的。电子传递产生穿过耦合膜的电化学质子梯度，并且质子沿着这些梯度流动通过ATP合酶驱动ATP合成。叶绿体ATP合酶含有9种不同的多肽，总共约20条多肽链。 该酶可以被分成两部分：CF 1和CFo。 CF 1含有酶的核苷酸结合位点，具有催化作用。 CFo将CF 1锚定在膜上并使质子移位。 将研究CF 1和CFo的多肽亚基之间的相互作用，重点是CF 1的较小亚基（γ，δ和γ）和活性的调节。 本项目将研究核苷酸结合在调控中的作用。CF 1内的e亚基的C-末端结构域的位置及其在ATP合成，调节和质子门控中的作用将进行评估，使广泛使用的定点突变。将研究CF 1中g亚基的C-末端部分的位置是否不同于线粒体F1中的位置。由于CF 1易于大量制备、能够调控其亚基组成和核苷酸含量以及类囊体的高ATP合成活性，叶绿体ATP合成酶成为本研究的极好对象。 这项工作的结果将适用于所有的ATP酶和蛋白质-蛋白质相互作用，一般。

Adenosine 5'-triphosphate (ATP) synthesis from adenosine 5'-diphosphate (ADP) and inorganic phosphate (Pi) is catalyzed by closely related enzymes in the energy transducing membranes of chloroplasts, mitochondria and some bacteria. Electron transport generates electrochemical proton gradients across the coupling membranes and the flow of protons down these gradients through the ATP synthase drives ATP synthesis. The chloroplast ATP synthase contains nine different polypeptides and a total of about twenty polypeptide chains. The enzyme may be separated into two parts: CF1 and CFo. CF1 contains the nucleotide binding sites of the enzyme and is catalytic. CFo anchors CF1 to the membrane and translocates protons. Interactions among the polypeptide subunits of CF1 and CFo will be studied, with emphasis on the smaller subunits of CF1 (gamma, delta and epsilon) and the regulation of activity. This project will examine roles of the binding of nucleotides in regulation. The location of the C-terminal domain of the e subunit within CF1 and its roles in ATP synthesis, regulation and proton gating will be evaluated making extensive use of site-directed mutagenesis. Whether the position of the C-terminal portion of the g subunit in CF1 differs from that in mitochondrial F1 will be investigated. The ease of preparation of large amounts of CF1, ability to manipulate its subunit composition and nucleotide content, and the high ATP synthesis activity of thylakoids, make the chloroplast ATP synthase an excellent subject for this research. The results of this work will be applicable to all ATP synthases and to protein-protein interactions, in general.