Oligomerization of the tetramerization domain of p53 probed by two- and three-color single-molecule FRET.

Abstract:

: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.

authors

Chung HS,Meng F,Kim JY,McHale K,Gopich IV,Louis JM

doi

10.1073/pnas.1700357114

subject

Has Abstract

pub_date

2017-08-15 00:00:00

pages

E6812-E6821

issue

33

eissn

0027-8424

issn

1091-6490

pii

1700357114

journal_volume

114

pub_type

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