Characterization of conformational changes and noncovalent complexes of myoglobin by electrospray ionization mass spectrometry, circular dichroism and fluorescence spectroscopy
Characterization of conformational changes and noncovalent complexes of myoglobin by electrospray ionization mass spectrometry, circular dichroism and fluorescence spectroscopy
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通过电喷雾电离质谱、圆二色性和荧光光谱法表征肌红蛋白的构象变化和非共价复合物
DOI：
10.1002/jms.1747
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发表时间：
2010-06-01
影响因子：
2.3
通讯作者：
Wang, Xian
中科院分区：
文献类型：
--
作者：
Lin, Xin;Zhao, Wenjie;Wang, Xian
Electrospray ionization mass spectrometry (ESI-MS) was employed to monitor the heme release and the conformational changes of myoglobin (Mb) under different solvent conditions, and to observe ligand bindings of Mb. ESI-MS, complemented by circular dichroism and fluorescence spectroscopy, was used to study the mechanism of acid- and organic solvent-induced denaturation by probing the changes in the secondary and the tertiary structure of Mb. The results obtained show that complete disruption of the heme-protein interactions occurs when Mb is subjected to one of the following solution conditions: pH 3.2-3.6, or solution containing 20-30% acetonitrile or 40-50% methanol. Outside these ranges, Mb is present entirely in its native state (binding with a heme group) or as apomyoglobin (i.e. without the heme). Spectroscopic data demonstrate that the denaturation mechanism of Mb induced by acid may be significantly different from that by the organic solvent. Low pH reduces helices in Mb, whereas certain organic content level in solution results in the loss of the tertiary structure. ESI-MS conditions were established to observe the H2O- and CO-bound Mb complexes, respectively. H2O binding to metmyoglobin (17 585 Da), where the heme iron is in the ferric oxidation state, is observed in ESI-MS. CO binding to Mb (17 595 Da), on the other hand, can be only observed after the heme iron is reduced to the ferrous form. Therefore, ESI-MS combined with spectroscopic techniques provides a useful means for probing the formation of ligand-binding complexes and characterizing protein conformational changes. Copyright (C) 2010 John Wiley & Sons, Ltd.
