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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.

Intrinsically-disordered proteins (IDPs) are unfolded at the native condition and fold only when attaching to their binding partners. Even though IDPs often undergo large conformational changes, which may slow the binding process, the binding kinetics are sometimes surprisingly fast, close to those of diffusion-controlled reactions. IDPs usually contain a large number of charged residues and theory predicts that the association can be facilitated by the formation of a transient complex (TC) stabilized by the electrostatic attraction between an IDP and its binding target. Although the modulation of the association rate by ionic strength of the solution has been observed for a number of IDPs, TC has not been directly probed during the course of binding. To experimentally investigate the role of TC in fast binding, we employed single-molecule Forster resonance energy transfer (FRET) spectroscopy that monitors the conformational evolution of an IDP from the unbound state to the bound complex. We studied binding of the transactivation domain (TAD) of the tumor suppressor protein p53 (negatively charged, −10) and the nuclear co-activator binding domain (NCBD) of CBP (positively charged, +6). The N- and C-termini of TAD were labeled with Alexa 647 and Alexa 488 site-specifically and molecules were immobilized on a glass surface using a biotin-neutravidin linkage to monitor binding to unlabeled NCBD in solution. Because of the fast binding kinetics, the bound state, TC, and unbound state could not be clearly resolved in binned trajectories. Instead of binning, we used the maximum likelihood method (Gopich and Szabo, JPCB(2009)) to analyze photon trajectories collected at high illumination intensity and to measure the binding kinetics (photon count rate of 50 - 100 ms−1) and the lifetime of TC (photon count rate of 500 - 1000 ms−1). We found that the association rate reaches ∼ 1 x 109 M−1s−1, which is close to the diffusion-limited rate, and the lifetime of TC is relatively long (∼100 microsecond) compared to the kinetics at low ionic strength (∼ 0.04 M). More importantly, both quantities decrease significantly with the increasing ionic strength, indicating that the destabilization of TC results in the slow association. This result demonstrates that the fast association of an IDP can be achieved by stabilizing TC through native/non-native electrostatic interactions, which would allow longer time for an IDP to interact with a target protein to fold and bind.

Bending with high curvature is one of the major mechanical properties of double-stranded DNA (dsDNA) for its biological functions. Local-melting in the middle of dsDNA (kink), which reduce the energy cost of bending, have been suggested as alternative DNA conformations in addition to the simple bending of dsDNA in the presence of high constrain force. However, the conformations of deformed dsDNA by high bending force and their dynamic characters remain unknown. Here, we report that the strong bending induces not only the kink in the middle of dsDNA but also the end-melting of dsDNA by applying single-molecule fluorescence resonance energy transfer (smFRET) to D-shaped DNA nanostructure consisted of dsDNA (30 bp) and single-stranded DNA (4 - 30 nt). We directly proved that two deformed structures of dsDNA are not permanent but dynamically interconverted each other in millisecond scales. The transition from end-melting to kink is dominated by entropy (anti-Arrhenius behavior), while the transition from kink to end-melting is dominated by enthalpy. The presence of the mismatch or permanent bubble in dsDNA accelerates the kink formation with less compressive force and the kink state becomes permanent when the size of permanent bubble is larger than three base pairs.

Using a single-molecule method, we directly distinguish among oligomers from monomers to tetramers and determine their quaternary structures. Using this method, we found that RecR forms a stable dimer and its oligomeric form is modulated by its own concentration and the interaction with RecO.