Pederson Kari, Chalmers Gordon R, Gao Qi, Elnatan Daniel, Ramelot Theresa A, Ma Li-Chung, Montelione Gaetano T, Kennedy Michael A, Agard David A, Prestegard James H
Complex Carbohydrate Research Center, University of Georgia, Athens, USA.
Department of Computer Science, University of Georgia, Athens, USA.
J Biomol NMR. 2017 Jul;68(3):225-236. doi: 10.1007/s10858-017-0123-8. Epub 2017 Jun 26.
A strategy for acquiring structural information from sparsely isotopically labeled large proteins is illustrated with an application to the E. coli heat-shock protein, HtpG (high temperature protein G), a 145 kDa dimer. It uses C-alanine methyl labeling in a perdeuterated background to take advantage of the sensitivity and resolution of Methyl-TROSY spectra, as well as the backbone-centered structural information from H-C residual dipolar couplings (RDCs) of alanine methyl groups. In all, 40 of the 47 expected crosspeaks were resolved and 36 gave RDC data. Assignments of crosspeaks were partially achieved by transferring assignments from those made on individual domains using triple resonance methods. However, these were incomplete and in many cases the transfer was ambiguous. A genetic algorithm search for consistency between predictions based on domain structures and measurements for chemical shifts and RDCs allowed 60% of the 40 resolved crosspeaks to be assigned with confidence. Chemical shift changes of these crosspeaks on adding an ATP analog to the apo-protein are shown to be consistent with structural changes expected on comparing previous crystal structures for apo- and complex- structures. RDCs collected on the assigned alanine methyl peaks are used to generate a new solution model for the apo-protein structure.
通过对大肠杆菌热休克蛋白HtpG（高温蛋白G，一种145 kDa的二聚体）的应用，阐述了一种从稀疏同位素标记的大蛋白中获取结构信息的策略。该策略在全氘代背景下使用C-丙氨酸甲基标记，以利用甲基-TROSY谱的灵敏度和分辨率，以及来自丙氨酸甲基基团的H-C剩余偶极耦合（RDC）的以主链为中心的结构信息。总共47个预期的交叉峰中有40个得到解析，36个给出了RDC数据。交叉峰的归属部分是通过将使用三重共振方法在各个结构域上做出的归属进行转移来实现的。然而这些归属并不完整，而且在许多情况下转移是不明确的。基于结构域结构的预测与化学位移和RDC测量值之间一致性的遗传算法搜索，使得40个解析的交叉峰中有60%能够得到可靠归属。向脱辅基蛋白中添加ATP类似物时这些交叉峰的化学位移变化，与比较脱辅基和复合物结构的先前晶体结构时预期的结构变化一致。在已归属的丙氨酸甲基峰上收集的RDC用于生成脱辅基蛋白结构的新溶液模型。
