Structural Evaluation of Protein/Metal Complexes via Native Electrospray Ultraviolet Photodissociation Mass Spectrometry
Structural Evaluation of Protein/Metal Complexes via Native Electrospray Ultraviolet Photodissociation Mass Spectrometry
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DOI：
10.1021/jasms.0c00066
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发表时间：
2020-05-06
影响因子：
3.2
通讯作者：
Brodbelt, Jennifer S.
中科院分区：
文献类型：
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作者：
Crittenden, Christopher M.;Novelli, Elisa T.;Brodbelt, Jennifer S.
紫外光解离（UVPD）已成为一种很有前景的工具，不仅可用于表征蛋白质的一级序列和翻译后修饰，还可用于表征其三级结构。在这项研究中，使用了三种金属结合蛋白——葡萄球菌核酸酶、天青蛋白和钙调蛋白，通过比较脱辅基（无金属）蛋白和全蛋白（金属结合）的裂解模式，展示了UVPD用于阐明金属结合区域的用途。除了评估葡萄球菌核酸酶与钙的结合外，还评估了一系列预计以与钙相似方式结合的镧系（III）离子。基于对UVPD光谱的比较分析，确定钙和镧系离子的结合区域从第40 - 50位残基延伸，与已知的晶体结构相符。对天青蛋白（探究铜和银的结合）和钙调蛋白（四个钙结合位点）也进行了类似的分析。这项工作证明了UVPD方法在确定和分析各类蛋白质金属结合位点方面的实用性。
Ultraviolet photodissociation (UVPD) has emerged as a promising tool to characterize proteins with regard to not only their primary sequences and post-translational modifications, but also their tertiary structures. In this study, three metal-binding proteins, Staphylococcal nuclease, azurin, and calmodulin, are used to demonstrate the use of UVPD to elucidate metal-binding regions via comparisons between the fragmentation patterns of apo (metal-free) and holo (metal-bound) proteins. The binding of staphylococcal nuclease to calcium was evaluated, in addition to a series of lanthanide(III) ions which are expected to bind in a similar manner as calcium. On the basis of comparative analysis of the UVPD spectra, the binding region for calcium and the lanthanide ions was determined to extend from residues 40-50, aligning with the known crystal structure. Similar analysis was performed for both azurin (interrogating copper and silver binding) and calmodulin (four calcium binding sites). This work demonstrates the utility of UVPD methods for determining and analyzing the metal binding sites of a variety of classes of proteins.