Abstract

Disulfide bridges are essential for maintaining the structure and function of proteins. Traditionally, studies of the disulfide bonds require expensive equipment and high purity of the protein sample, therefore, the development of simpler techniques is warranted. Here, were present a novel protocol for the detection of disulfide bonds in proteins, which is based on the labeling reduced disulfide bridges with a high molecular weight (HMW) maleimide derivative. After irreversible blocking of free thiol groups of proteins, the labeling of new thiols released from disulfide bridges with a high-molecular-weight (HMW) maleimide derivative is performed. To confirm localization of cysteines involved in the formation of disulfide bonds, cysteine mutagenesis was conducted. For validation, aquaporin 5 (AQP5) and transient receptor potential cation channel subfamily V member 4 (TRPV4) proteins were tagged with FLAG (DYKDDDDK) on N-termini. Increase in MW of the target proteins from immunoblot indicated the presence of disulfide bonds. No bands with increased MW were detected in AQP5, while TPRV4 cysteines at disulfide bridges-constituting positions 639, 645, 652, 660, 770 were detected and confirmed by cysteine mutagenesis. These data indicate that the proposed technique is feasible and effective for the detection of protein disulfide bonds.

中文翻译：

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通过标记高分子量马来酰亚胺衍生物检测到的蛋白质二硫键

摘要

二硫键对于维持蛋白质的结构和功能至关重要。传统上，对二硫键的研究需要昂贵的设备和蛋白质样品的高纯度，因此，有必要开发更简单的技术。在这里，提出了一种新的协议，用于检测蛋白质中的二硫键，该协议基于具有高分子量（HMW）马来酰亚胺衍生物的还原二硫键标记。不可逆地阻断蛋白质的游离硫醇基团后，用高分子量（HMW）马来酰亚胺衍生物标记从二硫键释放的新硫醇。为了证实参与二硫键形成的半胱氨酸的定位，进行了半胱氨酸诱变。为了验证，将水通道蛋白5（AQP5）和瞬时受体潜在阳离子通道亚家族V成员4（TRPV4）蛋白在N-末端上用FLAG（DYKDDDDK）标记。免疫印迹中靶蛋白分子量的增加表明存在二硫键。在AQP5中未检测到分子量增加的条带，而通过半胱氨酸诱变检测并确认了位于二硫键构成639、645、652、660、770的TPRV4半胱氨酸。这些数据表明，所提出的技术对于检测蛋白质二硫键是可行和有效的。TPRV4半胱氨酸位于二硫键构成的位置639、645、652、660、770，并通过半胱氨酸诱变得到确认。这些数据表明，所提出的技术对于检测蛋白质二硫键是可行和有效的。TPRV4半胱氨酸位于二硫键构成的位置639、645、652、660、770，并通过半胱氨酸诱变得到确认。这些数据表明，所提出的技术对于蛋白质二硫键的检测是可行和有效的。