Surface-entropy reduction used in the crystallization of human choline acetyltransferase
Surface-entropy reduction used in the crystallization of human choline acetyltransferase
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DOI：
10.1107/s0907444905018822
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发表时间：
2005-09-01
影响因子：
2.2
通讯作者：
Shilton, BH
中科院分区：
文献类型：
--
作者：
Kim, AR;Dobransky, T;Shilton, BH
人胆碱乙酰转移酶（ChAT）从胆碱和乙酰辅酶A合成神经递质乙酰胆碱（ACh）。人ChAT的晶体结构一直是神经元信号传导领域的长期目标。可纯化毫克量的纯ChAT [Kim等人（2005），Protein Expr.普里夫40，107 - 117]，但是穷尽的结晶努力未能产生任何适合于高分辨率结构研究的晶体。为了获得高质量的人ChAT晶体，在一个大的保守性差的环区域进行截短，并从蛋白质表面去除高熵侧链。所得的“熵降低的”ChAT（MR = 68.1kDa）容易且可重复地结晶，并且晶体将X射线散射至约2.2埃。这些晶体的可用性将使我们能够研究人类ChAT自身的结构以及与其底物和抑制剂分子的复合物，从而更好地了解其催化机制和调节。
Human choline acetyltransferase ( ChAT) synthesizes the neurotransmitter acetylcholine (ACh) from choline and acetyl-CoA. A crystal structure of human ChAT has been a long-standing goal in the neuronal signalling field. Milligram quantities of pure ChAT can be purified [Kim et al. ( 2005), Protein Expr. Purif. 40, 107 - 117], but exhaustive crystallization efforts failed to produce any crystals suitable for high-resolution structural studies. To obtain high-quality crystals of human ChAT, a truncation was made in a large poorly conserved loop region and high-entropy side chains were removed from the surface of the protein. The resulting 'entropy-reduced' ChAT (MR = 68.1 kDa) crystallizes readily and reproducibly and the crystals diffract X-rays to approximately 2.2 angstrom. The availability of these crystals will allow us to study the structure of human ChAT on its own as well as in complex with its substrates and inhibitor molecules, leading to a greater understanding of its catalytic mechanism and regulation.