Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870, USA.
J Biol Chem. 2011 May 27;286(21):18938-48. doi: 10.1074/jbc.M111.234609. Epub 2011 Apr 4.
Mass spectrometry-based hydrogen/deuterium exchange (H/DX) has been used to define the polypeptide backbone dynamics of full-length methyl CpG binding protein 2 (MeCP2) when free in solution and when bound to unmethylated and methylated DNA. Essentially the entire MeCP2 polypeptide chain underwent H/DX at rates faster than could be measured (i.e. complete exchange in ≤10 s), with the exception of the methyl DNA binding domain (MBD). Even the H/DX of the MBD was rapid compared with that of a typical globular protein. Thus, there is no single tertiary structure of MeCP2. Rather, the full-length protein rapidly samples many different conformations when free in solution. When MeCP2 binds to unmethylated DNA, H/DX is slowed several orders of magnitude throughout the MBD. Binding of MeCP2 to methylated DNA led to additional minor H/DX protection, and only locally within the N-terminal portion of the MBD. H/DX also was used to examine the structural dynamics of the isolated MBD carrying three frequent mutations associated with Rett syndrome. The effects of the mutations ranged from very little (R106W) to a substantial increase in conformational sampling (F155S). Our H/DX results have yielded fine resolution mapping of the structure of full-length MeCP2 in the absence and presence of DNA, provided a biochemical basis for understanding MeCP2 function in normal cells, and predicted potential approaches for the treatment of a subset of RTT cases caused by point mutations that destabilize the MBD.
基于质谱的氢/氘交换（H/DX）已被用于定义游离状态下全长甲基 CpG 结合蛋白 2（MeCP2）和与非甲基化及甲基化 DNA 结合时的多肽骨架动力学。基本上，除了甲基化 DNA 结合域（MBD）之外，整个 MeCP2 多肽链的 H/DX 速度都快于可测量的速度（即在≤10s 内完全交换）。即使是 MBD 的 H/DX 也比典型的球状蛋白快。因此，MeCP2 没有单一的三级结构。相反，当游离在溶液中时，全长蛋白质会迅速地采样许多不同的构象。当 MeCP2 与非甲基化 DNA 结合时，MBD 中的 H/DX 会被减缓几个数量级。MeCP2 与甲基化 DNA 的结合导致 MBD 内的局部区域出现额外的轻微 H/DX 保护作用。H/DX 还被用于研究携带与雷特综合征相关的三种常见突变的分离 MBD 的结构动力学。突变的影响范围从很小（R106W）到构象采样的显著增加（F155S）。我们的 H/DX 结果提供了在存在和不存在 DNA 的情况下全长 MeCP2 结构的精细分辨率图谱，为理解正常细胞中 MeCP2 功能提供了生化基础，并预测了通过点突变导致 MBD 不稳定的 RTT 病例的潜在治疗方法。
