Chen J, Smith D L
Department of Chemistry and Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska, Lincoln, Nebraska 68588-0304, USA.
Biochemistry. 2000 Apr 18;39(15):4250-8. doi: 10.1021/bi992619n.
The chaperonin GroEL is a homotetradecamer in which the subunits (M(r) 57 000) are joined through noncovalent forces. This study reports on the unfolding and disassembly of GroEL in guanidine hydrochloride and urea. Kinetic and equilibrium measurements were made using amide hydrogen exchange/mass spectrometry, light scattering, and size-exclusion chromatography. Hydrogen exchange in GroEL destabilized in 1.8 M GdHCl (the unfolding midpoint is 1.2 M GdHCl) shows that the apical and intermediate domains unfold 3.1 times faster than the equatorial domain. Light scattering measurements made under the same conditions show that disassembly of the native GroEL tetradecamer occurs at the same rate as unfolding of the equatorial domain. This study of the kinetics of GroEL unfolding and disassembly demonstrates the existence of an intermediate that was identified as a tetradecamer with the apical and intermediate domains unfolded. Although this intermediate was easily detected in dynamic unfolding measurements, its population in equilibrium measurements at the midpoint for GroEL unfolding was too small to be detected. This study of GroEL unfolding and disassembly points to features that may be important in the folding and assembly of the GroEL macroassembly.
伴侣蛋白GroEL是一种同十四聚体，其亚基（相对分子质量57 000）通过非共价力结合在一起。本研究报道了GroEL在盐酸胍和尿素中的解折叠和拆卸情况。使用酰胺氢交换/质谱、光散射和尺寸排阻色谱进行了动力学和平衡测量。在1.8 M盐酸胍中不稳定的GroEL中的氢交换（解折叠中点为1.2 M盐酸胍）表明，顶端和中间结构域的解折叠速度比赤道结构域快3.1倍。在相同条件下进行的光散射测量表明，天然GroEL十四聚体的拆卸速度与赤道结构域的解折叠速度相同。对GroEL解折叠和拆卸动力学的这项研究证明存在一种中间体，该中间体被鉴定为顶端和中间结构域已解折叠的十四聚体。尽管这种中间体在动态解折叠测量中很容易检测到，但在GroEL解折叠中点的平衡测量中其数量太少而无法检测到。对GroEL解折叠和拆卸的这项研究指出了在GroEL大分子组装的折叠和组装中可能重要的特征。
