Imidazole C-2 hydrogen/deuterium exchange reaction at histidine for probing protein structure and function with matrix-assisted laser desorption ionization mass spectrometry.
Imidazole C-2 hydrogen/deuterium exchange reaction at histidine for probing protein structure and function with matrix-assisted laser desorption ionization mass spectrometry.
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DOI：
10.1021/bi401260f
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发表时间：
2014-03-25
期刊：
影响因子：
2.9
通讯作者：
Nakazawa, Takashi
中科院分区：
文献类型：
--
作者：
Hayashi, Naoka;Kuyama, Hiroki;Nakajima, Chihiro;Kawahara, Kazuki;Miyagi, Masaru;Nishimura, Osamu;Matsuo, Hisayuki;Nakazawa, Takashi
我们提出了一种用于分析蛋白质结构和功能的质谱方法，该方法基于咪唑C-2或组氨酸Cε1氢/氘（H/D）交换反应，该反应相对于D2 O中咪唑阳离子和OD−的浓度本质上是二级的。该反应的二级速率常数（k2）根据伪一级速率常数（kφ）的pH依赖性计算，该伪一级速率常数（kφ）由孵育时间（t）时含有确定组氨酸残基的肽片段的平均质量ΔMr（0 ≤ ΔMr < 1）的变化获得，使得kφ = − [ln（1-ΔMr）]/t。我们更喜欢使用k2而不是kφ，因为 （k2的最大值）与pKa经验相关，如Brønsted图所示： （α是一个任意常数），因此我们可以分析结构对H/D交换率的影响， 表示k2与。在牛核糖核酸酶A的催化位点中，His 12显示出更大的变化， 与His 119相比，与胞苷3′-单磷酸结合后，正如从X射线结构和溶剂可及性的可能变化所预期的那样。然而，需要考虑咪唑基团与不可解离基团的氢键来解释His 48在部分溶剂暴露的情况下的极慢的H/D交换速率。
We present a mass spectrometric method for analyzing protein structure and function, based on the imidazole C-2 or histidine Cε1 hydrogen/deuterium (H/D) exchange reaction, which is intrinsically second order with respect to the concentrations of the imidazolium cation and OD− in D2O. The second-order rate constant (k2) of this reaction was calculated from the pH-dependency of the pseudo-first-order rate constant (kφ) obtained from the change of average mass ΔMr (0 ≤ ΔMr < 1) of a peptide fragment containing a defined histidine residue at incubation time (t) such that kφ = − [ln(1−ΔMr)]/t. We preferred using k2 rather than kφ because (maximal value of k2) was empirically related to pKa as illustrated with a Brønsted plot: (α is an arbitrary constant), so that we could analyze the effect of structure on the H/D-exchange rate in terms of representing the deviation of k2 from . In the catalytic site of bovine ribonuclease A, His12 showed much larger change in compared with His119 upon binding with cytidine 3′-monophosphate, as anticipated from the X-ray structures and the possible change in solvent accessibility. However, there is a need of considering the hydrogen bonds of the imidazole group with non-dissociable groups to interpret an extremely slow H/D exchange rate of His48 in partially solvent-exposed situation.
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影响因子：
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作者：
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