SITES OF PROTEIN PROTEIN-INTERACTION ON THE MITOCHONDRIAL F1-ATPASE INHIBITOR PROTEIN
SITES OF PROTEIN PROTEIN-INTERACTION ON THE MITOCHONDRIAL F1-ATPASE INHIBITOR PROTEIN
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DOI：
10.1042/bj2350577
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发表时间：
1986-04-15
影响因子：
4.1
通讯作者：
HARRIS, DA
中科院分区：
文献类型：
--
作者：
JACKSON, PJ;HARRIS, DA
用差示示踪法研究了牛心线粒体F1-ATP酶抑制剂蛋白的结构。这种方法也被用来确定位点的抑制剂蛋白参与结合分离的线粒体ATP酶（F1）和一个特定的抗抑制剂抗体。天然的游离抑制剂用[14 C]乙酸酐在其赖氨酸和丝氨酸残基上进行示踪标记，并且抑制剂蛋白质在氯化胍中展开或用[3 H]乙酸酐特异性结合至另一种蛋白质。通过h.p.l.c.分离后，从抑制剂蛋白水解消化物中肽的14 C/3 H比率推断残基的暴露/隐藏。天然抑制剂中的赖氨酸或丝氨酸残基都不像未折叠形式那样暴露。存在反应性梯度，其中残基54-58最隐蔽，并且暴露朝向蛋白质的任一末端增加。在残基1-3中观察到反应性轻微降低，表明N-末端可能处于相当受限的环境中。这些研究结果进行了讨论，在光的抑制剂蛋白的预测结构。当抑制蛋白与F1结合时，除一个标记残基外的所有标记残基的反应性都增加。例外的是Lys-24，只是稍微隐藏了一些。因此，F1结合似乎不直接涉及赖氨酸或丝氨酸残基。这一发现与F1-抑制剂相互作用本质上是疏水性的观点一致。使用抗抑制剂抗体提供补充信息，该抗体与抑制剂上不同于F1结合的位点结合。该抗体的结合相当大地隐藏了残基54、58和65。这证实了F1不与抑制剂蛋白上的这些亲水性残基相互作用。
We have investigated the structure of the mitochondrial F1-ATPase inhibitor protein from ox heart by using a differential trace-labelling method. This method has also been used to determine sites on the inhibitor protein involved in binding both the isolated mitochondrial ATPase (F1) and to a specific anti-inhibitor antibody. Native, free inhibitor was trace-labelled on its lysine and serine residues with [14C]acetic anhydride, and inhibitor protein unfolded in guanidinium chloride or specifically bound to another protein, with [3H]acetic anhydride. Exposure/concealment of residues was deduced from the 14C/3H ratios of the peptides in a proteolytic digest of the inhibitor, after separation by h.p.l.c. None of the lysine or serine residues in the native inhibitor are as exposed as in the unfolded form. There is a gradient of reactivity, with residues 54-58 being most concealed and exposure increasing towards either end of the protein. A slight decrease in reactivity is noted in residues 1-3, suggesting that the N-terminus may be in a fairly restricted environment. These findings are discussed in the light of the predicted structure of the inhibitor protein. All but one of the labelled residues increases in reactivity when inhibitor protein binds to F1. The exception, Lys-24, is only slightly concealed. Hence, F1 binding appears not to involve the lysine or serine residues directly. This finding is consistent with the view that the F1-inhibitor interaction is hydrophobic in nature. Complementary information was provided using an anti-inhibitor antibody that binds to a site on the inhibitor different from that at which F1 binds. Binding of this antibody conceals residues 54, 58 and 65 considerably. This confirms that F1 does not interact with these hydrophilic residues on the inhibitor protein.