Local dynamics measured by hydrogen/deuterium exchange and mass spectrometry of creatine kinase digested by two proteases
Local dynamics measured by hydrogen/deuterium exchange and mass spectrometry of creatine kinase digested by two proteases
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DOI：
10.1016/j.biochi.2005.05.012
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发表时间：
2005-12-01
期刊：
影响因子：
3.9
通讯作者：
Vial, C
中科院分区：
文献类型：
--
作者：
Mazon, H;Marcillat, O;Vial, C
氢/氘交换耦合质谱法研究了天然二聚体胞质肌酸激酶的结构和动力学。蛋白在D2O中孵育不同时间。经过H/D交换和快速猝灭反应后，部分氘化蛋白被两种不同的蛋白酶（胃蛋白酶或来自曲霉的XIII型蛋白酶）平行切割，以增加氘融合的序列覆盖率和空间分辨率。所得多肽采用液相色谱-质谱联用分析。与MM-CK的三维结构相比，与先前使用胃蛋白酶进行的研究相比，对两种独立的蛋白水解氘化模式的分析使我们对CK局部动力学有了新的认识[Mazon等]。蛋白质科学13(2004)476-486]。特别是，我们获得了更多关于以1-22和362-380胃蛋白酶肽为代表的N端和c端氘交换动力学和程度的信息。事实上，我们观察到1-12和13-22型XIII蛋白酶肽的行为非常不同，362-373和374-380肽也有类似的行为。此外，通过比较90-126型胃蛋白酶肽的XIII型蛋白酶段的氘化模式，我们确定了一个较小的相对动态区域（108-114）。(C) 2005 Elsevier SAS。版权所有。
Hydrogen/deuterium exchange coupled to mass spectrometry has been used to investigate the structure and dynamics of native dimeric cytosolic muscle creatine kinase. The protein was incubated in D2O for various time. After H/D exchange and rapid quenching of the reaction, the partially deuterated protein was cleaved in parallel by two different proteases (pepsin or type XIII protease from Aspergillus saitoi) to increase the sequence coverage and spatial resolution of deuterium incorporation. The resulting peptides were analyzed by liquid chromatography coupled to mass spectrometry. In comparison with the 3D structure of MM-CK, the analysis of the two independent proteolysis deuteration patterns allowed us to get new insights into CK local dynamics as compared to a previous study using pepsin [Mazon et al. Protein Science 13 (2004) 476-486]. In particular, we obtained more information on the kinetics and extent of deuterium exchange in the N- and C-terminal extremities represented by the 1-22 and 362-380 pepsin peptides. Indeed, we observed a very different behaviour of the 1-12 and 13-22 type XIII protease peptides, and similarly for the 362-373 and 374-380 peptides. Moreover, comparison of the deuteration patterns of type XIII protease segments of the large 90-126 pepsin peptide led us to identify a small relatively dynamic region (108-114). (C) 2005 Elsevier SAS. All rights reserved.