Gorovits B M, Horowitz P M
Department of Biochemistry, University of Texas Health Science Center at San Antonio 78284, USA.
J Biol Chem. 1995 Dec 1;270(48):28551-6. doi: 10.1074/jbc.270.48.28551.
The urea-induced dissociation and subsequent conformational transitions of the nucleotide-bound form of GroEL were studied by light scattering, 4,4'-bis(1-anilino-8- naphthalenesulfonic acid) binding, and intrinsic tyrosine fluorescence. Magnesium ion alone (10 mM) stabilizes GroEL and leads to coordination of the structural transitions monitored by the different parameters. The midpoint of the light-scattering transition that monitored dissociation of the 14-mer with bound magnesium was raised to approximately 3 M, which is considerably higher than the ligand-free form of the protein, which exhibits a transition with a midpoint at approximately 2 M urea. Binding of ADP results in destabilization of the GroEL oligomeric structure, and complete dissociation of the 14-mer in the presence of 5 mM ADP occurs at about 2 M urea with the midpoint of the transition at approximately 1 M urea. The same destabilization by ADP and stabilization by Mg2+ were seen when the conformation was followed by the intrinsic fluorescence. Complexation with the nonhydrolyzable ATP analog, 5'-adenylimidodiphosphate gave an apparent stability of the quaternary structure that was between that observed with Mg2+ and that with ADP. The ADP-bound form of the protein demonstrated increased hydrophobic exposure at lower urea concentrations than the uncomplexed GroEL. In addition, the GroEL-ADP complex is more accessible for proteolytic digestion by chymotrypsin than the uncomplexed protein, consistent with a more open, flexible form of the protein. The implication of the conformational changes to the mechanism of the GroEL function is discussed.
通过光散射、4,4'-双（1-苯胺基-8-萘磺酸）结合以及内在酪氨酸荧光，研究了尿素诱导的与核苷酸结合形式的GroEL的解离及随后的构象转变。单独的镁离子（10 mM）可稳定GroEL，并导致由不同参数监测的结构转变的协同作用。监测与结合镁的14聚体解离的光散射转变的中点提高到约3 M，这大大高于无配体形式的蛋白质，其在约2 M尿素时呈现中点的转变。ADP的结合导致GroEL寡聚体结构不稳定，在5 mM ADP存在下，14聚体在约2 M尿素时完全解离，转变中点在约1 M尿素。当通过内在荧光跟踪构象时，观察到ADP产生的相同去稳定作用和Mg2+产生的稳定作用。与不可水解的ATP类似物5'-腺苷亚氨基二磷酸络合，使四级结构具有明显的稳定性，介于用Mg2+和用ADP观察到的稳定性之间。与未复合的GroEL相比，蛋白质的ADP结合形式在较低尿素浓度下表现出增加的疏水暴露。此外，与未复合的蛋白质相比，GroEL-ADP复合物更容易被胰凝乳蛋白酶进行蛋白水解消化，这与蛋白质更开放、更灵活的形式一致。讨论了构象变化对GroEL功能机制的影响。
