Stockmar Florian, Kobitski Andrei Yu, Nienhaus Gerd Ulrich
Institute of Applied Physics, Karlsruhe Institute of Technology (KIT) , Wolfgang-Gaede-Strasse 1, 76131 Karlsruhe, Germany.
Institute of Nanotechnology, Karlsruhe Institute of Technology (KIT) , Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany.
J Phys Chem B. 2016 Feb 4;120(4):641-9. doi: 10.1021/acs.jpcb.5b09336. Epub 2016 Jan 21.
We have studied the folding kinetics of the core intermediate (I) state of RNase H by using a combination of single-molecule FRET (smFRET) and hidden Markov model analysis. To measure fast dynamics in thermal equilibrium as a function of the concentration of the denaturant GdmCl, a special FRET labeled variant, RNase H 60-113, which is sensitive to folding of the protein core, was immobilized on PEGylated surfaces. Conformational transitions between the unfolded (U) state and the I state could be described by a two-state model within our experimental time resolution, with millisecond mean residence times. The I state population was always a minority species in the entire accessible range of denaturant concentrations. By introducing the measured free energy differences between the U and I states as constraints in global fits of the GdmCl dependence of FRET histograms of a differently labeled RNase H variant (RNase H 3-135), we were able to reveal the free energy differences and, thus, population ratios of all three macroscopic state ensembles, U, I and F (folded state) as a function of denaturant concentration.
我们通过结合单分子荧光共振能量转移（smFRET）和隐马尔可夫模型分析，研究了核糖核酸酶H核心中间体（I）状态的折叠动力学。为了测量热平衡状态下作为变性剂盐酸胍（GdmCl）浓度函数的快速动力学，一种对蛋白质核心折叠敏感的特殊荧光共振能量转移标记变体核糖核酸酶H 60-113被固定在聚乙二醇化表面上。在我们的实验时间分辨率范围内，未折叠（U）状态和I状态之间的构象转变可以用双态模型来描述，平均停留时间为毫秒级。在变性剂浓度的整个可及范围内，I状态群体始终是少数物种。通过将测量得到的U状态和I状态之间的自由能差作为约束条件，纳入另一种标记的核糖核酸酶H变体（核糖核酸酶H 3-135）的FRET直方图对GdmCl依赖性的全局拟合中，我们能够揭示所有三个宏观状态集合U、I和F（折叠状态）的自由能差，进而得到它们作为变性剂浓度函数的群体比例。
