Yang Haopeng, Xiao Ming, Wang Yuhong
University of Houston, 4800 Calhoun Rd, Houston, TX 77214, USA.
University of Houston, 4800 Calhoun Rd, Houston, TX 77214, USA.
Biophys Chem. 2015 Apr;199:39-45. doi: 10.1016/j.bpc.2015.02.008. Epub 2015 Mar 3.
The unique advantage of the single molecule approach is to reveal the inhomogeneous subpopulations in an ensemble. For example, smFRET (single molecule fluorescence resonance energy transfer) can identify multiple subpopulations based on the FRET efficiency histograms. However, identifying multiple FRET states with overlapping average values remains challenging. Here, we report a new concept and method to analyze the single molecule FRET data of a ribosome system. The main results are as follows: 1. based on a hierarchic concept, multiple ribosome subpopulations are identified. 2. The subpopulations are self-identified via the cross-correlation analysis of the FRET histogram profiles. The dynamic heterogeneity is tracked after 2 min intervals on the same ribosomes individually. 3. The major ribosome subpopulations exchange with each other with a certain pattern, indicating some correlations among the motions of the tRNAs and the ribosomal components. Experiments under the conditions of 20% glycerol or 1mM viomycin supported this conclusion.
单分子方法的独特优势在于能够揭示总体中的非均匀亚群。例如，单分子荧光共振能量转移（smFRET）可基于FRET效率直方图识别多个亚群。然而，识别具有重叠平均值的多个FRET状态仍然具有挑战性。在此，我们报告一种用于分析核糖体系统单分子FRET数据的新概念和方法。主要结果如下：1.基于层次概念，识别出多个核糖体亚群。2.通过FRET直方图谱的互相关分析自动识别亚群。在相同核糖体上每隔2分钟单独追踪动态异质性。3.主要核糖体亚群以特定模式相互交换，表明tRNA和核糖体组分的运动之间存在一些相关性。在20%甘油或1mM紫霉素条件下的实验支持了这一结论。
