Hunter C L, Mauk A G, Douglas D J
Protein Engineering Network of Centres of Excellence, University of British Columbia, Vancouver, Canada.
Biochemistry. 1997 Feb 4;36(5):1018-25. doi: 10.1021/bi961993+.
The relationship of the structure of a protein in solution to the structure of a gas-phase protein ion and the manner in which gas-phase protein ions bind small molecules noncovalently are topics of current debate. To address these issues, the stability of heme binding to wild-type and variant forms of apomyoglobin and apocytochrome b5 has been studied in the gas phase by electrospray mass spectrometry (ES-MS) and compared with the stability of heme binding to the same proteins in solution. The voltage required to dissociate ions of the heme-protein complexes in the orifice-skimmer region of an electrospray mass spectrometer, a measure of the complex stability, is found to be correlated with the activation energy for dissociation of the complexes in solution across a series of proteins in which the number of hydrogen bonds between the heme propionate groups and surface residues is systematically reduced. However, variants in which the hydrogen bonds to the proximal histidine have been removed are destabilized in solution but stabilized in the gas-phase ions. These results suggest that on the millisecond time scale of the ES-MS experiment, the gas-phase protein ion may retain much of the structure of the protein in solution, at least for those residues surrounding the heme group. Furthermore, the ability of ES-MS to detect relatively subtle differences in protein-small molecule complex stability demonstrated in this work suggests that this technique may be a convenient, sensitive, and generally useful strategy for physical characterization of such complexes.
溶液中蛋白质的结构与气相蛋白质离子的结构之间的关系，以及气相蛋白质离子非共价结合小分子的方式，是当前争论的话题。为了解决这些问题，通过电喷雾质谱（ES-MS）在气相中研究了血红素与脱辅基肌红蛋白和脱辅基细胞色素b5的野生型及变体形式结合的稳定性，并与血红素与这些蛋白质在溶液中结合的稳定性进行了比较。发现在电喷雾质谱仪的孔口-分离器区域中使血红素-蛋白质复合物离子解离所需的电压（一种复合物稳定性的度量），与一系列蛋白质中复合物在溶液中解离的活化能相关，在这些蛋白质中，血红素丙酸基团与表面残基之间的氢键数量被系统地减少。然而，与近端组氨酸的氢键已被去除的变体在溶液中不稳定，但在气相离子中稳定。这些结果表明，在ES-MS实验的毫秒时间尺度上，气相蛋白质离子可能保留了溶液中蛋白质的大部分结构，至少对于血红素基团周围的那些残基是如此。此外，这项工作中ES-MS检测蛋白质-小分子复合物稳定性相对细微差异的能力表明，该技术可能是用于此类复合物物理表征的一种方便、灵敏且普遍有用的策略。
