Study on the regulatory region of the chloroplast ATP synthase by using peptide antibody

为了阐明叶绿体三磷酸腺苷合成酶的调控机制，我们采用了三种不同的方法：(1)γ亚基调节区的突变分析，(2)硫氧还蛋白与γ亚基调节区的相互作用的研究，(3)硫氧还蛋白与叶绿体三磷酸腺苷合成酶F1部分相互作用的研究。我们首先研究了在γ亚基上的两个半胱氨酸的调节区周围引入突变对调控的影响。从这项研究中，我们得出结论，调控区域本身的构象变化对酶活性的调节是最关键的。此外，我们还发现调控区域负电荷簇的缺失导致了调控的逆转。其次，我们制备了突变的硫氧还蛋白来研究其与ATP合成酶的相互作用。通过本研究，我们可以阐明叶绿体硫氧还蛋白仅通过蛋白质-蛋白质相互作用才能诱导γ亚基的重要构象变化，从而促进调节性二硫键的还原。虽然我们制备了针对叶绿体硫氧还蛋白活性区域的抗体来研究硫氧还蛋白与2ATP合成酶的相互作用，但对这一方向的研究还不成功。

To clarify the regulatory mechanism of the chloroplast ATP synthase, we carried out three different approach as follows ; (1) the mutation analysis of the regulatory region of the γ subunit, (2) the investigation of the interaction between thioredoxin and the regulatory region of the γ subunit, (3) the investigation of the interaction between thioredoxin and the chloroplast ATP synthase F_1 part.We first investigated the change of the regulation by the introduction of the mutation at around the regulatory region including two cysteines on the γ subunit. From this study, we concluded that the conformational change of the regulatory region itself is the most critical for the regulation of the enzyme activity. Furthermore, we found that the deletion of the negative charged cluster in the regulatory region results the inversion of the regulation.Secondary, we prepared the mutant thioredoxin to investigate the interaction with ATP synthase. From this study, we could clarify that the chloroplast thioredoxin can induce the important conformational change of the γ subunit to facilitate the reduction of the regulatory disulfide bond only by the protein-protein interaction. Although we prepared the antibody against the active region of the chloroplast thiredoxin to investigate the interaction between thioredoxin and 2ATP synthase, the study on this direction is not successful yet.