A back hydrogen exchange procedure via the acid-unfolded state for a large protein.
A back hydrogen exchange procedure via the acid-unfolded state for a large protein.
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DOI：
10.1021/bi300495p
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发表时间：
2012-07
期刊：
影响因子：
2.9
通讯作者：
Mototaka Suzuki;K. Sakurai;Young-Ho Lee;T. Ikegami;K. Yokoyama;Y. Goto
中科院分区：
文献类型：
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作者：
Mototaka Suzuki;K. Sakurai;Young-Ho Lee;T. Ikegami;K. Yokoyama;Y. Goto
大分子蛋白质的核磁共振(核磁共振)测量需要氢化蛋白质样品，因为高分子量会导致严重的信号展宽。用(2)H(2)O表示的氚样品随后应在酰胺基上进行反向氢交换。为了进行反向交换，蛋白质分子被展开或不稳定，这样内部残基就可以被溶剂接触到。然而，大蛋白质在不稳定或未折叠状态下的复性产率通常很低，这导致了大蛋白质的核磁共振测量的两难境地。在我们以前的论文中[Suzuki，M.，et al.(2011年)生物化学50,10390-10398]，我们提出了在低盐条件下可以有效地回收由331个氨基酸残基组成的酸变性微生物谷氨酰胺转氨酶，从而避开了容易聚集的中间体。在这里，我们证明了ProMTG，由376个氨基酸残基组成的Pro形式的MTG，可以在低盐条件下从酸未折叠的状态完美复性，如圆二色谱和核磁共振光谱所证实的。通过对(2)H(2)O中表达的氢代ProMTG执行相同的过程，我们通过核磁共振光谱观察到内部残基的完全反向交换。我们的程序具有潜在的应用于大蛋白质的背氢交换的核磁共振测量。
A deuterated protein sample is required for nuclear magnetic resonance (NMR) measurements of a large protein because severe signal broadenings occur because of the high molecular weight. The deuterated sample expressed in (2)H(2)O should subsequently be subjected to a back hydrogen exchange at amide groups. To perform the back exchange, the protein molecule is unfolded or destabilized so that internal residues become accessible to the solvent. However, the refolding yield from the destabilized or unfolded state of a large protein is usually low, leading to a dilemma in NMR measurements of large proteins. In our previous paper [Suzuki, M., et al. (2011) Biochemistry 50, 10390-10398], we suggested that an acid-denatured microbial transglutaminase (MTG) consisting of 331 amino acid residues can be recovered effectively under low-salt conditions, escaping from the aggregation-prone intermediate. Here, we demonstrate that proMTG, the pro form of MTG consisting of 376 amino acid residues, can be refolded perfectly from the acid-unfolded state under low-salt conditions, as confirmed by circular dichroism and NMR spectroscopies. By performing the same procedure with a deuterated proMTG expressed in (2)H(2)O, we observed complete back exchanges for internal residues by NMR spectroscopy. Our procedure has potential applications to the back hydrogen exchange of large proteins for NMR measurements.