Higurashi T, Nosaka K, Mizobata T, Nagai J, Kawata Y
Department of Biotechnology Faculty of Engineering, Tottori University, Tottori, 680-0945, Japan.
J Mol Biol. 1999 Aug 20;291(3):703-13. doi: 10.1006/jmbi.1999.2994.
The guanidine-hydrochloride (Gdn-HCl) induced unfolding and refolding characteristics of the co-chaperonin GroES from Escherichia coli, a homoheptamer of subunit molecular mass 10,000 Da, were studied by using intrinsic fluorescence, 1-anilino-8-naphthalene sulfonate (ANS) binding, and size-exclusion HPLC. When monitored by tyrosine fluorescence, the unfolding reaction of GroES consisted of a single transition, with a transition midpoint at around 1.0 M Gdn-HCl. Interestingly, however, ANS binding and size-exclusion HPLC experiments strongly suggested the existence of an intermediate state in the transition. In order to confirm the existence of an intermediate state between the native heptameric and unfolded monomeric states, a tryptophan residue was introduced into the interface of GroES subunits as a fluorescent probe. The unfolding reaction of GroES I48W as monitored by tryptophyl fluorescence showed a single transition curve with a transition midpoint at 0.5 M Gdn-HCl. This unfolding transition curve as well as the refolding kinetics were dependent on the concentration of GroES protein. CD spectrum and size-exclusion HPLC experiments demonstrated that the intermediates assumed a partially folded conformation at around 0.5 M Gdn-HCl. The refolding of GroES protein from 3 M Gdn-HCl was probed functionally by measuring the extent of inhibition of GroEL ATPase activity and the enhancement of lactate dehydrogenase refolding yields in the presence of GroEL and ADP. These results clearly demonstrated that the GroES heptamer first dissociated to monomers and then unfolded completely upon increasing the concentration of Gdn-HCl, and that both transitions were reversible. From the thermodynamic analysis of the dissociation reaction, it was found that the partially folded monomer was only marginally stable and that the stability of GroES protein is governed mostly by the association of the subunits.
采用内源荧光、1-苯胺基-8-萘磺酸盐（ANS）结合及尺寸排阻高效液相色谱法，研究了来自大肠杆菌的共伴侣蛋白GroES（亚基分子量为10,000 Da的同型七聚体）在盐酸胍（Gdn-HCl）诱导下的去折叠和重折叠特性。当通过酪氨酸荧光监测时，GroES的去折叠反应由单一转变组成，转变中点在约1.0 M Gdn-HCl处。然而，有趣的是，ANS结合和尺寸排阻高效液相色谱实验强烈表明在该转变过程中存在中间状态。为了证实天然七聚体状态和去折叠单体状态之间存在中间状态，将一个色氨酸残基作为荧光探针引入GroES亚基的界面。通过色氨酸荧光监测的GroES I48W的去折叠反应显示出单一的转变曲线，转变中点在0.5 M Gdn-HCl处。该去折叠转变曲线以及重折叠动力学均取决于GroES蛋白的浓度。圆二色光谱和尺寸排阻高效液相色谱实验表明，中间体在约0.5 M Gdn-HCl时呈现部分折叠构象。通过测量在GroEL和ADP存在下GroEL ATP酶活性的抑制程度以及乳酸脱氢酶重折叠产率的提高，从功能上探究了GroES蛋白从3 M Gdn-HCl的重折叠情况。这些结果清楚地表明，随着Gdn-HCl浓度的增加，GroES七聚体首先解离为单体，然后完全去折叠，并且这两个转变都是可逆的。从解离反应的热力学分析发现，部分折叠的单体仅略微稳定，并且GroES蛋白的稳定性主要由亚基的缔合决定。
