Analysis of the solution conformations of T4 lysozyme by paramagnetic NMR spectroscopy
Analysis of the solution conformations of T4 lysozyme by paramagnetic NMR spectroscopy
复制标题
通过顺磁核磁共振波谱分析 T4 溶菌酶的溶液构象。
DOI：
10.1039/c5cp07196h
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发表时间：
2016-02-28
影响因子：
3.3
通讯作者：
Otting, Gottfried
中科院分区：
文献类型：
--
作者：
Chen, Jia-Liang;Yang, Yin;Otting, Gottfried
噬菌体T4溶菌酶（T4-L）的大量晶体结构表明，它含有两个亚结构域，它们可以排列成紧凑的构象（闭合状态），或者在T4-L的突变体中，更延伸的结构（开放状态）。在溶液中，野生型T4-L仅显示单组核磁共振（NMR）信号，掩盖了任何构象异质性。为了探测T4-L的构象空间，我们通过将4-巯基甲基吡啶二羧酸通过二硫键连接到T4-L的三突变体C54 T/C97 A/S44 C中的Cys 44来产生位点特异性镧系元素结合位点，并测量假接触位移（PCS）和磁诱导残余偶极耦合（RDC）。数据表明，在溶液中并且在不存在底物的情况下，T4-L的结构平均比野生型T4-L的晶体结构的闭合构象所暗示的更开放。通过假设涉及更开放的构象和封闭状态的人口加权的两态模型获得了稍微改进的拟合，但是用Gd 3+测量的顺磁弛豫增强反对这样的构象平衡。拟合不能通过包括从T4-L突变体的数百种晶体结构中挑选的第三种构象来改善。
A large number of crystal structures of bacteriophage T4 lysozyme (T4-L) have shown that it contains two subdomains, which can arrange in a compact conformation (closed state) or, in mutants of T4-L, more extended structures (open state). In solution, wild-type T4-L displays only a single set of nuclear magnetic resonance (NMR) signals, masking any conformational heterogeneity. To probe the conformational space of T4-L, we generated a site-specific lanthanide binding site by attaching 4-mercaptomethyl dipicolinic acid via a disulfide bond to Cys44 in the triple-mutant C54T/C97A/S44C of T4-L and measured pseudocontact shifts (PCS) and magnetically induced residual dipolar couplings (RDC). The data indicate that, in solution and in the absence of substrate, the structure of T4-L is on average more open than suggested by the closed conformation of the crystal structure of wild-type T4-L. A slightly improved fit was obtained by assuming a population-weighted two-state model involving an even more open conformation and the closed state, but paramagnetic relaxation enhancements measured with Gd3+ argue against such a conformational equilibrium. The fit could not be improved by including a third conformation picked from the hundreds of crystal structures available for T4-L mutants.