Subunit and Nucleotide Interactions in the Chloroplast ATP Synthase

腺苷5′-二磷酸（ADP）和无机磷酸（Pi）合成腺苷5′-三磷酸（ATP）是由叶绿体、线粒体和一些细菌的能量转导膜上密切相关的酶催化的。电子传递在偶联膜上产生电化学质子梯度，质子流通过ATP合酶沿着这些梯度流动，驱动ATP合成。叶绿体ATP合酶包含9种不同的多肽，总共约有20条多肽链。酶可分为两部分：CF1和CFo。CF1含有该酶的核苷酸结合位点，具有催化作用。CFo将CF1固定在膜上并转运质子。CF1和CFo多肽亚基之间的相互作用将被研究，重点是CF1较小的亚基（γ， δ和epsilon）和活性调节。本项目将研究核苷酸结合在调控中的作用。CF1中e亚基的c端结构域的位置及其在ATP合成、调控和质子门控中的作用将通过广泛使用定点诱变来评估。g亚基在CF1中的c端位置是否与线粒体F1中的位置不同将被研究。由于CF1易于大量制备，可控制其亚基组成和核苷酸含量，以及类囊体的高ATP合成活性，使叶绿体ATP合酶成为本研究的理想对象。总的来说，这项工作的结果将适用于所有ATP合酶和蛋白质-蛋白质相互作用。

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.