Local Folding and Misfolding in the PBX Homeodomain from a Three-State Analysis of CPMG Relaxation Dispersion NMR Data
Local Folding and Misfolding in the PBX Homeodomain from a Three-State Analysis of CPMG Relaxation Dispersion NMR Data
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DOI：
10.1021/jp306127m
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发表时间：
2012-08-30
影响因子：
3.3
通讯作者：
Mittermaier, Anthony K.
中科院分区：
文献类型：
--
作者：
Farber, Patrick J.;Slager, Jelle;Mittermaier, Anthony K.
核磁共振Carr-Purcell-Meiom-Gill(CPMG)弛豫弥散实验是表征蛋白质内部运动和深入了解蛋白质折叠、催化和变构等基本生物过程的有效方法。在大多数情况下，CPMG数据的分析假设蛋白质在两个不同的构象状态之间交换。用CPMG核磁共振表征在两个以上状态之间交换的体系更具挑战性。例如，在快时间尺度下的三态交换的情况下，很难将从CPMG分析中提取的参数与最感兴趣的物理参数、互换率、布居和交换态的化学位移差唯一地联系起来。我们开发了一种网格搜索选择程序，允许根据CPMG数据唯一地确定这些物理参数，在本研究中，这些信息包括配体诱导的化学位移扰动。我们将这种方法应用于PBX同源结构域(PBX-HD)，这是一种带有C末端延伸的三螺旋蛋白质，在与DNA结合时折叠成第四螺旋。我们最近发现，即使在没有DNA的情况下，C-末端的延伸也是瞬时折叠的，这一过程可能与PBX-HD与DNA和其他同源结构域的协同结合有关。使用网格搜索选择程序，我们发现PBX-HD经历了三种不同的构象状态之间的交换，即C-末端延伸被展开的主要形式，C-末端延伸形成第四螺旋的先前确定的状态，以及C-末端延伸错误折叠的附加状态。
NMR Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments represent a powerful approach for characterizing protein internal motions and for gaining insight into fundamental biological processes such as protein folding, catalysis, and allostery. In most cases, CPMG data are analyzed assuming that the protein exchanges between two different conformational states. Systems exchanging among more than two states are far more challenging to characterize by CPMG NMR. For example, in the case of three-state exchange in the fast time scale regime, it is difficult to uniquely connect the parameters extracted from CPMG analyses with the physical parameters of most interest, intercoversion rates, populations, and chemical shift differences for exchanging states. We have developed a grid search selection procedure that allows these physical parameters to be uniquely determined from CPMG data, based on additional information, which in this study comprises ligand-induced chemical shift perturbations. We applied this approach to the PBX homeodomain (PBX-HD), a three-helix protein with a C-terminal extension that folds into a fourth helix upon binding to DNA. We recently showed that the C-terminal extension transiently folds, even in the absence DNA, in a process that is likely tied to the cooperative binding of PBX-HD to DNA and other homeodomains. Using the grid search selection procedure, we found that PBX-HD undergoes exchange between three different conformational states, a major form in which the C-terminal extension is unfolded, the previously identified state in which the C-terminal extension forms a fourth helix, and an additional state in which the C-terminal extension is misfolded.