Tang Chun, Ghirlando Rodolfo, Clore G Marius
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA.
J Am Chem Soc. 2008 Mar 26;130(12):4048-56. doi: 10.1021/ja710493m. Epub 2008 Mar 4.
Ultra-weak macromolecular self-association is exceptionally difficult to both detect and visualize using conventional biophysical techniques owing to the very low population of the associated species, yet such weak intermolecular interactions coupled with nucleation events play an important role in driving spontaneous self-assembly to form higher-order architectures (such as crystals, viral capsids, and amyloid fibrils). In this article, we detect and characterize transient, ultra-weak self-association (KD >or= 15 mM) involving the histidine-containing protein HPr by means of paramagnetic relaxation enhancement (PRE), using EDTA-Mn2+ conjugated at three separate sites (E5C, E25C, and E32C, one at a time). Large intermolecular PRE effects, above the background observed with hydroxylamine-EDTA-Mn2+ as a control, are observed for two of the three paramagnetically labeled sites, E5C and E32C. The extent of self-association can be modulated (significantly reduced) by increasing the ionic strength or by the introduction of a negative charge (S46D mutation) within a positively charged surface patch, and abolished upon the addition of the N-terminal domain of enzyme I (EIN) to form a specific EIN-HPr complex. The PRE profiles observed for E5C and E32C can be fitted simultaneously and accounted for quantitatively using conjoined rigid body/torsion angle dynamics-simulated annealing with an ensemble of states to represent the distribution of one molecule of HPr relative to its partner.
由于相关物种的数量极低，使用传统生物物理技术检测和可视化超弱大分子自缔合异常困难，然而这种弱分子间相互作用与成核事件在驱动自发自组装形成高阶结构（如晶体、病毒衣壳和淀粉样纤维）中起着重要作用。在本文中，我们通过顺磁弛豫增强（PRE），使用在三个不同位点（E5C、E25C和E32C，每次一个位点）共轭的EDTA-Mn²⁺，检测并表征了涉及含组氨酸蛋白HPr的瞬时超弱自缔合（KD≥15 mM）。对于三个顺磁标记位点中的两个，即E5C和E32C，观察到了大于以羟胺-EDTA-Mn²⁺作为对照所观察到的背景的大分子PRE效应。自缔合程度可以通过增加离子强度或在带正电的表面斑块内引入负电荷（S46D突变）来调节（显著降低），并且在添加酶I（EIN）的N端结构域以形成特定的EIN-HPr复合物后被消除。观察到的E5C和E32C的PRE谱可以同时拟合，并使用连体刚体/扭转角动力学模拟退火结合一组状态来定量解释，以表示一个HPr分子相对于其伙伴的分布。
