Department of Chemistry, California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, California 94720, USA.
Biochemistry. 2011 Sep 27;50(38):8251-60. doi: 10.1021/bi200640s. Epub 2011 Aug 30.
The technique of hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) has been applied to a mesophilic (E. coli) dihydrofolate reductase under conditions that allow direct comparison to a thermophilic (B. stearothermophilus) ortholog, Ec-DHFR and Bs-DHFR, respectively. The analysis of hydrogen-deuterium exchange patterns within proteolytically derived peptides allows spatial resolution, while requiring a series of controls to compare orthologous proteins with only ca. 40% sequence identity. These controls include the determination of primary structure effects on intrinsic rate constants for HDX as well as the use of existing 3-dimensional structures to evaluate the distance of each backbone amide hydrogen to the protein surface. Only a single peptide from the Ec-DHFR is found to be substantially more flexible than the Bs-DHFR at 25 °C in a region located within the protein interior at the intersection of the cofactor and substrate-binding sites. The surrounding regions of the enzyme are either unchanged or more flexible in the thermophilic DHFR from B. stearothermophilus. The region with increased flexibility in Ec-DHFR corresponds to one of two regions previously proposed to control the enthalpic barrier for hydride transfer in Bs-DHFR [Oyeyemi et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 10074].
氢氘交换结合质谱（HDX-MS）技术已应用于中温（大肠杆菌）二氢叶酸还原酶，条件允许与嗜热（嗜热脂肪芽孢杆菌）同源物 Ec-DHFR 和 Bs-DHFR 进行直接比较。在蛋白水解衍生肽内分析氢氘交换模式可以实现空间分辨率，同时需要进行一系列对照，以比较仅有约 40%序列同一性的同源蛋白。这些对照包括确定对 HDX 固有速率常数的一级结构效应，以及利用现有三维结构评估每个骨架酰胺氢与蛋白质表面的距离。在 25°C 下，仅在位于辅因子和底物结合位点交叉处的蛋白质内部区域中，从 Ec-DHFR 发现一个肽段比 Bs-DHFR 显著更具柔韧性。酶的周围区域在嗜热脂肪芽孢杆菌的嗜热 DHFR 中要么保持不变，要么更具柔韧性。在 Ec-DHFR 中增加柔韧性的区域与先前提出的控制 Bs-DHFR 中氢化物转移焓障碍的两个区域之一相对应[Oyeyemi 等人（2010 年）Proc. Natl. Acad. Sci. U.S.A. 107, 10074]。
