Multi-Color Single Molecule FRET Study of Intrinsically Disordered Protein Binding

Intrinsically disordered proteins (IDPs) are unstructured at the native condition and fold when attaching to their binding partners. Understanding the mechanism of this process requires probing conformational changes of IDPs during binding processes. Since multiple binding pathways should exist as protein folding, single-molecule spectroscopy is expected to provide unique information such as the distribution of binding pathways. In order to probe conformational changes of IDPs and their interactions with binding targets simultaneously, it is necessary to obtain the distance information between more than two fluorophores. In this work, we performed three-color FRET spectroscopy to study the oligomerization of the tetramerization domain (TD) of the tumor suppressor protein p53. Two monomers of TD form a dimer at low nM concentration and subsequently two dimers form a tetramer at higher concentration. In the dimerization experiment, one monomer TD construct was labeled with Alexa 488 and Alexa 647 and immobilized on a PEG-coated glass coverslip via a biotin-streptavidin linkage. Another TD construct was labeled with Alexa 750 as a binding partner in solution. Using the alternating excitation of two picosecond-pulsed lasers (485 and 640 nm) at 40 MHz, it was possible to detect all three FRET efficiencies between three fluorophores. In addition, from the average delay times between photon arrivals and laser excitation, the fluorescence lifetime of each state was measured. We determined the dissociation constant and the dissociation rate of the dimer, which are consistent with the previous measurements by fluorescence correlation spectroscopy. More importantly, we observed TD monomer conformations in the dimer that are different from that in the tetramer. The faster formation of the tetramer than the dissociation of the dimer at high TD concentration suggests that these dimers with different conformations should be on pathway intermediates during the tetramerization.