Structures of invisible, excited protein states by relaxation dispersion NMR spectroscopy
Structures of invisible, excited protein states by relaxation dispersion NMR spectroscopy
复制标题
DOI：
10.1073/pnas.0804221105
复制
发表时间：
2008-08-19
影响因子：
11.1
通讯作者：
Kay, Lewis E.
中科院分区：
文献类型：
--
作者：
Vallurupalli, Pramodh;Hansen, D. Flemming;Kay, Lewis E.
分子的功能通常是预测在基态和更高的能量构象，可以发挥重要作用的配体结合，分子识别，酶催化和蛋白质折叠之间的偏移。结构生物学的工具使基态结构和动力学的详细表征;然而，激发态构象的研究更困难，因为它们的人口少，可能只存在短暂的。在这里，我们描述了一种基于弛豫色散NMR光谱的方法，在该方法中，从化学位移和残余各向异性磁相互作用获得不可见的激发态的结构。为了建立该方法的实用性，我们研究了交换蛋白质（Abp 1 p SH 3结构域）-配体（Ark 1 p肽）系统，其中肽仅以少量添加，使得配体结合形式不可见。根据N-15、（HN）-H-1、C-13（α）和（CO）-C-13化学位移的集合，以及（HN）-H-1-N-15、H-1（α）-C-13（α）和（HN）-H-1-（CO）-C-13残余偶极耦合和（CO）-C-13残余化学位移各向异性，所有这些都与不可见的结合构象有关，确定了结合态的结构。通过与在第二配向介质中记录的（HN）-H-1-N-15残余偶极偶联比较，交叉验证如此获得的结构。所描述的方法打开了详细的结构研究的可能性，在迄今为止一直局限于应用程序涉及可见的基态蛋白质的细节水平的无形蛋白质构象。
Molecular function is often predicated on excursions between ground states and higher energy conformers that can play important roles in ligand binding, molecular recognition, enzyme catalysis, and protein folding. The tools of structural biology enable a detailed characterization of ground state structure and dynamics; however, studies of excited state conformations are more difficult because they are of low population and may exist only transiently. Here we describe an approach based on relaxation dispersion NMR spectroscopy in which structures of invisible, excited states are obtained from chemical shifts and residual anisotropic magnetic interactions. To establish the utility of the approach, we studied an exchanging protein (Abp1p SH3 domain)-ligand (Ark1p peptide) system, in which the peptide is added in only small amounts so that the ligand-bound form is invisible. From a collection of N-15, (HN)-H-1, C-13(alpha), and (CO)-C-13 chemical shifts, along with (HN)-H-1-N-15, H-1(alpha)-C-13(alpha), and (HN)-H-1-(CO)-C-13 residual dipolar couplings and (CO)-C-13 residual chemical shift anisotropies, all pertaining to the invisible, bound conformer, the structure of the bound state is determined. The structure so obtained is cross-validated by comparison with (HN)-H-1-N-15 residual dipolar couplings recorded in a second alignment medium. The methodology described opens up the possibility for detailed structural studies of invisible protein conformers at a level of detail that has heretofore been restricted to applications involving visible ground states of proteins.