Solvent Mimicry with Methylene Carbene to Probe Protein Topography
Solvent Mimicry with Methylene Carbene to Probe Protein Topography
复制标题
DOI：
10.1021/acs.analchem.5b02724
复制
发表时间：
2015-10-06
影响因子：
7.4
通讯作者：
Delfino, Jose Maria
中科院分区：
文献类型：
--
作者：
Gomez, Gabriela Elena;Monti, Jose Luis E.;Delfino, Jose Maria
多肽链的溶剂可及表面积（SASA）在蛋白质折叠、构象变化和相互作用中起着关键作用。这个基本的生物物理参数在实验测量中是难以捉摸的。我们解决这个问题的方法依赖于最小的光化学试剂二氮丙啶（DZN）与多肽的反应。该试剂（1）发挥溶剂模拟作用，因为它的大小与水相当，和（ii）显示很少的化学选择性，因为它产生极其反应性的亚甲基卡宾。甲基化产生EM（修饰程度）信号，其可用于仔细检查由Ca 2+与钙调蛋白（CaM）结合引发的构象变化。对于全蛋白观察到的增加的EM由Ca 2 +-CaM中疏水区域的增强的暴露主导。片段化使我们能够量化在特定位点的亚甲基掺入。肽91-106揭示了钙151结合位点周围的主要重组，导致局部有序和疏水表面的更大暴露。此外，该技术显示出对CaM和蜂毒肽（Mel）之间的探针识别的高灵敏度。EM的大幅降低表明络合后显著疏水区域的闭塞。保护标签揭示了一个更大的参与N-末端和中央区域的钙调素在这种相互作用。尽管其规模较小，梅尔的差异暴露也可以量化。此外，MS/MS片段化实现了在氨基酸水平上扩展标记位点分辨率的目标。总的来说，DZN标记作为一种有用的足迹法出现，能够揭示生理构象变化和相互作用。
The solvent accessible surface area (SASA) of the polypeptide chain plays a key role in protein folding, conformational change, and interaction. This fundamental biophysical parameter is elusive in experimental measurement. Our approach to this problem relies on the reaction of the minimal photochemical reagent diazirine (DZN) with polypeptides. This reagent (1) exerts solvent mimicry because its size is comparable to water and (ii) shows scant chemical selectivity because it generates extremely reactive methylene carbene. Methylation gives rise to the EM (extent of modification) signal, which is useful for scrutinizing the conformational change triggered by Ca2+ binding to calmodulin (CaM). The increased EM observed for the full protein is dominated by the enhanced exposure of hydrophobic area in Ca2+-CaM. Fragmentation allowed us to quantify the methylene incorporation at specific sites. Peptide 91-106 reveals a major reorganization around the calcium 151 binding site, resulting in local ordering and a greater exposure of the hydrophobic surface. Additionally, this technique shows a high sensitivity to probe recognition between CaM and melittin (Mel). The large decrease in EM indicates the occlusion of a significant hydrophobic area upon complexation. Protection from labeling reveals a larger involvement of the N-terminal and central regions of CaM in this interaction. Despite its smaller size, Mel's differential exposure can also be quantified. Moreover, MS/MS fragmentation realizes the goal of extending the resolution of labeled sites at the amino acid level. Overall, DZN labeling emerges as a useful footprinting method capable of shedding light on physiological conformational changes and interactions.