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A back hydrogen exchange procedure via the acid-unfolded state for a large protein

机译：通过大分子蛋白质的酸展开状态进行反向氢交换程序

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摘要

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 ~2H _2O 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) Biochemistry50, 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 ~2H _2O, 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.
机译：大蛋白质的核磁共振（NMR）测量需要氘代蛋白质样品，因为高分子量会导致严重的信号展宽。 〜2H _2O中表达的氘代样品应随后在酰胺基团上进行反向氢交换。为了进行反向交换，使蛋白质分子解折叠或不稳定，以使内部残基易于接触溶剂。但是，大蛋白不稳定或未折叠状态的重折叠收率通常很低，导致大蛋白的NMR测量陷入困境。在我们以前的论文中[Suzuki，M.，et al。 （2011）Biochemistry50，10390-10398]，我们建议，由331个氨基酸残基组成的酸变性微生物转谷氨酰胺酶（MTG）可在低盐条件下有效地回收，避免从易于聚集的中间体中逸出。在这里，我们证明了proMTG（由376个氨基酸残基组成的MTG的前身）可以在低盐条件下从酸解折叠状态完美折叠，如圆二色性和NMR光谱学所证实。通过用〜2H _2O中表达的氘代proMTG执行相同的步骤，我们通过NMR光谱观察到内部残基的完全反向交换。我们的程序对大分子蛋白质的氢交换反应进行NMR测量具有潜在的应用前景。

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