Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.
J Am Chem Soc. 2010 Feb 3;132(4):1222-3. doi: 10.1021/ja909466d.
The histone acetyltransferase (HAT) p300/CBP has been shown to undergo autoacetylation on lysines in an apparent regulatory loop that stimulates HAT activity. Here we have developed a strategy to introduce acetyl-Lys at up to six known modification sites in p300/CBP HAT using a combination of circular permutation and expressed protein ligation. We show that these semisynthetic, circularly permuted acetylated proteins retain high affinity for an acetyl-CoA substrate analogue and that HAT activity correlates positively with degree of acetylation. This study provides novel evidence for control of p300/CBP HAT activity by site-specific autoacetylation and outlines a potentially general strategy for using expressed protein ligation and circular permutation to chemically interrogate internal regions of proteins.
组蛋白乙酰转移酶（HAT）p300/CBP 已被证明在一个明显的调节环中发生赖氨酸的自身乙酰化，这刺激了 HAT 活性。在这里，我们开发了一种策略，使用环化排列和表达蛋白连接的组合，在 p300/CBP HAT 中的多达六个已知修饰位点上引入乙酰-Lys。我们表明，这些半合成的、环化排列的乙酰化蛋白保留了与乙酰辅酶 A 类似物的高亲和力，并且 HAT 活性与乙酰化程度呈正相关。这项研究为 p300/CBP HAT 活性的特异性自身乙酰化控制提供了新的证据，并概述了一种使用表达蛋白连接和环化排列来化学探测蛋白质内部区域的潜在通用策略。
