文章基本信息

标题：Oligomerization of the tetramerization domain of p53 probed by two- and three-color single-molecule FRET
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作者：Hoi Sung Chung ; Fanjie Meng ; Jae-Yeol Kim 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2017
卷号：114
期号：33
页码：E6812-E6821
DOI：10.1073/pnas.1700357114
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：We describe a method that combines two- and three-color single-molecule FRET spectroscopy with 2D FRET efficiency–lifetime analysis to probe the oligomerization process of intrinsically disordered proteins. This method is applied to the oligomerization of the tetramerization domain (TD) of the tumor suppressor protein p53. TD exists as a monomer at subnanomolar concentrations and forms a dimer and a tetramer at higher concentrations. Because the dissociation constants of the dimer and tetramer are very close, as we determine in this paper, it is not possible to characterize different oligomeric species by ensemble methods, especially the dimer that cannot be readily separated. However, by using single-molecule FRET spectroscopy that includes measurements of fluorescence lifetime and two- and three-color FRET efficiencies with corrections for submillisecond acceptor blinking, we show that it is possible to obtain structural information for individual oligomers at equilibrium and to determine the dimerization kinetics. From these analyses, we show that the monomer is intrinsically disordered and that the dimer conformation is very similar to that of the tetramer but the C terminus of the dimer is more flexible.
关键词：single-molecule spectroscopy ; three-color FRET ; intrinsically disordered protein ; p53 oligomerization ; fluorescence lifetime