Histidine hydrogen-deuterium exchange mass spectrometry for probing the microenvironment of histidine residues in dihydrofolate reductase.
Histidine hydrogen-deuterium exchange mass spectrometry for probing the microenvironment of histidine residues in dihydrofolate reductase.
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DOI：
10.1371/journal.pone.0017055
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发表时间：
2011-02-16
期刊：
影响因子：
3.7
通讯作者：
Dealwis C
中科院分区：
文献类型：
--
作者：
Miyagi M;Wan Q;Ahmad MF;Gokulrangan G;Tomechko SE;Bennett B;Dealwis C
组氨酸氢-氘交换质谱法（His-HDX-MS）测定组氨酸残基中咪唑c2 -氢的HDX速率。该方法不仅提供了组氨酸咪唑环的HDX速率，而且还提供了组氨酸咪唑环的pK值。利用His-HDX-MS检测大肠杆菌二氢叶酸还原酶（DHFR）组氨酸残基的微环境，该酶在催化过程中发生多种构象变化。采用His-HDX-MS测定了载脂蛋白DHFR、与甲氨蝶呤络合DHFR （DHFR-MTX）、与MTX-NADPH络合DHFR （DHFR-MTX-NADPH）、与叶酸-NADP+络合DHFR （DHFR-叶酸-NADP+）的5种组氨酸残基在HDX反应中的pK值和半衰期（t1 /2）。结果表明，pK a和t1 /2对组氨酸残基周围微环境的变化较为敏感。尽管5个组氨酸残基中有4个远离活性位点，但配体结合影响了它们的pK a、t1 /2或两者都有。这与先前观察到的配体结合诱导的DHFR远端构象变化是一致的。大多数观察到的pK a和t1 /2的变化可以通过apo-DHFR， DHFR-MTX-NADPH和dhfr -叶酸- nadp +的x射线结构来解释。DHFR-MTX中子衍射结构的可用性使我们能够比较组氨酸咪唑环的质子化状态。我们的结果证明了His-HDX-MS在探测蛋白质中组氨酸残基的微环境中的有用性。
Histidine Hydrogen-Deuterium Exchange Mass Spectrometry (His-HDX-MS) determines the HDX rates at the imidazole C2-hydrogen of histidine residues. This method provides not only the HDX rates but also the pK a values of histidine imidazole rings. His-HDX-MS was used to probe the microenvironment of histidine residues of E. coli dihydrofolate reductase (DHFR), an enzyme proposed to undergo multiple conformational changes during catalysis. Using His-HDX-MS, the pK a values and the half-lives (t 1/2) of HDX reactions of five histidine residues of apo-DHFR, DHFR in complex with methotrexate (DHFR-MTX), DHFR in complex with MTX and NADPH (DHFR-MTX-NADPH), and DHFR in complex with folate and NADP+ (DHFR-folate-NADP+) were determined. The results showed that the two parameters (pK a and t 1/2) are sensitive to the changes of the microenvironment around the histidine residues. Although four of the five histidine residues are located far from the active site, ligand binding affected their pK a, t 1/2 or both. This is consistent with previous observations of ligand binding-induced distal conformational changes on DHFR. Most of the observed pK a and t 1/2 changes could be rationalized using the X-ray structures of apo-DHFR, DHFR-MTX-NADPH, and DHFR-folate-NADP+. The availability of the neutron diffraction structure of DHFR-MTX enabled us to compare the protonation states of histidine imidazole rings. Our results demonstrate the usefulness of His-HDX-MS in probing the microenvironments of histidine residues within proteins.
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影响因子：
56.9
作者：
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