Determination of Leu Side-Chain Conformations in Excited Protein States by NMR Relaxation Dispersion
Determination of Leu Side-Chain Conformations in Excited Protein States by NMR Relaxation Dispersion
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DOI：
10.1021/ja909294n
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发表时间：
2010-01-13
影响因子：
15
通讯作者：
Kay, Lewis E.
中科院分区：
文献类型：
--
作者：
Hansen, D. Flemming;Neudecker, Philipp;Kay, Lewis E.
拟合的Carr-Purcell-Meiboom-Gill（CPMG）弛豫色散曲线允许提取交换反应的动力学和热力学，交换反应使高度填充的基态和低填充的激发态构象相互转化。结构信息也可以以相互转换蛋白质状态之间的化学位移差异的形式获得。在这里，我们提出了一个非常简单的方法提取chi（2）旋转异构体分布的Leu侧链在'看不见的'激发态蛋白质的基础上测量其C-13（δ 1）/C-13（δ 2）的化学位移使用甲基CPMG分散实验。该方法被应用于研究Fyn SH 3结构域的蛋白质折叠反应。对于未折叠状态的Leu残基，获得了均匀的chi（2）旋转异构体分布，其中每个Leu以2：1的比例占据反式和侧边+构象。相比之下，“不可见的”Fyn SH 3结构域折叠中间体的亮氨酸显示出chi（2）旋转异构体群体的更不均匀的分布。该实验提供了一个重要的工具，对定量表征的结构和动力学性质的国家，不能研究其他生物物理工具。
Fits of Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion profiles allow extraction of the kinetics and thermodynamics of exchange reactions that interconvert highly populated, ground state and low Populated, excited state conformers. Structural information is also available in the form of chemical shift differences between the interconverting protein states. Here we present a very simple method for extracting chi(2) rotamer distributions of Leu side chains in 'invisible' excited protein states based on measurement of their C-13(delta 1)/C-13(delta 2) chemical shifts using methyl CPMG dispersion experiments. The methodology is applied to study the protein folding reaction of the Fyn SH3 domain. A uniform chi(2) rotamer distribution is obtained for Leu residues of the unfolded state, with each Leu occupying the trans and gauche+ conformations in a 2:1 ratio. By contrast, leucines of an 'invisible' Fyn SH3 domain folding intermediate show a much more heterogeneous distribution of chi(2) rotamer populations. The experiment provides an important tool toward the quantitative characterization of both the structural and dynamics properties of states that cannot be studied by other biophysical tools.