Folding pathway of FKBP12 and characterisation of the transition state
Folding pathway of FKBP12 and characterisation of the transition state
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DOI：
10.1006/jmbi.1999.2941
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发表时间：
1999-08-13
影响因子：
5.6
通讯作者：
Jackson, SE
中科院分区：
文献类型：
--
作者：
Main, ERG;Fulton, KF;Jackson, SE
人FKBP 12（一种12 kDa FK 506结合蛋白（亲免蛋白））的折叠途径已得到表征。展开和再折叠速率常数已被确定在广泛的变性剂浓度和数据显示，以适应折叠的两个状态模型，其中只有变性和原生状态显着填充，即使在没有变性剂。这种简单的折叠模型，其中没有中间状态显着填充，进一步支持停流圆二色性实验中没有快速的“突发”阶段观察。FKBP 12具有107个残基，是迄今为止最大的蛋白质，其在水中以简单的双态动力学折叠（k（F）= 4s（-1），25 ℃）。在FKBP 12中的两个环的拓扑交叉的结构元件，建议在折叠过程中引起动力学陷阱，似乎对折叠pathway.的过渡状态的折叠的影响不大，其特征在于野生型FKBP 12的一系列实验。的热力学性质，溶剂的可及性，和二级结构中，过渡态的信息，从实验测量的展开和折叠速率常数作为温度，变性剂浓度和三氟乙醇浓度的函数。此外，在配体存在下的解折叠和重折叠研究提供了过渡态配体结合口袋结构的信息。这些数据表明，一个紧凑的过渡态相对于未折叠的状态与一些70%的表面积埋。主要由两个环形成的配体结合位点在过渡态中基本上是非结构化的。三氟乙醇实验表明，α-螺旋可能在过渡态形成。这些结果与蛋白质工程研究和分子动力学模拟的结果进行了比较（见随附论文）。(C)北京：科学出版社.
The folding pathway of human FKBP12, a 12 kDa FK506-binding protein (immunophilin), has been characterised. Unfolding and refolding rate constants have been determined over a wide range of denaturant concentrations and data are shown to fit to a two-state model of folding in which only the denatured and native states are significantly populated, even in the absence of denaturant. This simple model for folding, in which no intermediate states are significantly populated, is further supported from stopped-flow circular dichroism experiments in which no fast "burst" phases are observed. FKBP12 with 107 residues, is the largest protein to date which folds with simple two-state kinetics in water (k(F) = 4 s(-1) at 25 degrees C). The topological crossing of two loops in FKBP12 a structural element suggested to cause kinetic traps during folding, seems to have little effect on the folding pathway.The transition state for folding has been characterised by a series of experiments on wild-type FKBP12. Information on the thermodynamic nature of, the solvent accessibility of, and secondary structure in, the transition state was obtained from experiments measuring the unfolding and refolding rate constants as a function of temperature, denaturant concentration and trifluoroethanol concentration. Ln addition, unfolding and refolding studies in the presence of Ligand provided information on the structure of the Ligand-binding pocket in the transition state. The data suggest a compact transition state relative to the unfolded state with some 70% of the surface area buried. The Ligand-binding site, which is formed mainly by two loops, is largely unstructured in the transition state. The trifluoroethanol experiments suggest that the a-helix may be formed in the transition state. These results are compared with results from protein engineering studies and molecular dynamics simulations (see the accompanying paper). (C) 1999 Academic Press.