Keller Bettina G, Kobitski Andrei, Jäschke Andres, Nienhaus G Ulrich, Noé Frank
Freie Universität Berlin , Institute of Chemistry and Biochemistry, Takustr. 3, 14195 Berlin, Germany.
J Am Chem Soc. 2014 Mar 26;136(12):4534-43. doi: 10.1021/ja4098719. Epub 2014 Mar 14.
We have developed a hidden Markov model and optimization procedure for photon-based single-molecule FRET data, which takes into account the trace-dependent background intensities. This analysis technique reveals an unprecedented amount of detail in the folding kinetics of the Diels-Alderase ribozyme. We find a multitude of extended (low-FRET) and compact (high-FRET) states. Five states were consistently and independently identified in two FRET constructs and at three Mg(2+) concentrations. Structures generally tend to become more compact upon addition of Mg(2+). Some compact structures are observed to significantly depend on Mg(2+) concentration, suggesting a tertiary fold stabilized by Mg(2+) ions. One compact structure was observed to be Mg(2+)-independent, consistent with stabilization by tertiary Watson-Crick base pairing found in the folded Diels-Alderase structure. A hierarchy of time scales was discovered, including dynamics of 10 ms or faster, likely due to tertiary structure fluctuations, and slow dynamics on the seconds time scale, presumably associated with significant changes in secondary structure. The folding pathways proceed through a series of intermediate secondary structures. There exist both compact pathways and more complex ones, which display tertiary unfolding, then secondary refolding, and, subsequently, again tertiary refolding.
我们针对基于光子的单分子荧光共振能量转移（FRET）数据开发了一种隐马尔可夫模型及优化程序，该程序考虑了与迹线相关的背景强度。这种分析技术揭示了狄尔斯-阿尔德酶核酶折叠动力学中前所未有的大量细节。我们发现了大量的伸展态（低FRET）和紧凑态（高FRET）。在两种FRET构建体以及三种Mg(2+)浓度下，一致且独立地识别出了五种状态。添加Mg(2+)后，结构通常趋于变得更加紧凑。观察到一些紧凑结构显著依赖于Mg(2+)浓度，这表明存在由Mg(2+)离子稳定的三级折叠。观察到一种紧凑结构与Mg(2+)无关，这与折叠后的狄尔斯-阿尔德酶结构中发现的三级沃森-克里克碱基配对导致的稳定性一致。发现了一个时间尺度层次结构，包括10毫秒或更快的动力学过程，这可能是由于三级结构波动引起的；以及秒级时间尺度上的慢动力学过程，大概与二级结构的显著变化有关。折叠途径通过一系列中间二级结构进行。既存在紧凑途径，也存在更复杂的途径，后者表现为三级结构展开，然后二级结构重新折叠，随后再次进行三级结构重新折叠。
