Single-Molecule characterization of oligomerization kinetics and equilibria of the tumor suppressor p53

Abstract

The state of oligomerization of the tumor suppressor p53 is an important factor in its various biological functions. It has a well-defined tetramerization domain, and the protein exists as monomers, dimers and tetramers in equilibrium. The dissociation constants between oligomeric forms are so low that they are at the limits of measurement by conventional methods in vitro. Here, we have used the high sensitivity of single-molecule methods to measure the equilibria and kinetics of oligomerization of full-length p53 and its isolated tetramerization domain, p53tet, at physiological temperature, pH and ionic strength using fluorescence correlation spectroscopy (FCS) in vitro. The dissociation constant at 37 °C for tetramers dissociating into dimers for full-length p53 was 50 ± 7 nM, and the corresponding value for dimers into monomers was 0.55 ± 0.08 nM. The half-lives for the two processes were 20 and 50 min, respectively. The equivalent quantities for p53tet were 150 ± 10 nM, 1.0 ± 0.14 nM, 2.5 ± 0.4 min and 13 ± 2 min. The data suggest that unligated p53 in unstressed cells should be predominantly dimeric. Single-molecule FCS is a useful procedure for measuring dissociation equilibria, kinetics and aggregation at extreme sensitivity.

Figure 1.

Kinetic stability of p53 studied using FCS. (A) Linear representation of p53 domain organization. (B) ACFs of WT-flp53, T-flp53 and T-flp53-Y220C measured after 15 min. (C) Aggregation kinetics of WT-flp53 and T-flp53-Y220C. (D) Aggregation kinetics of super-stable mutant T-flp53 and p53tet.

Figure 2.

Dissociation kinetics and equilibrium constants of p53tet oligomerization measured using FCS. (A) Normalized ACFs following the dissociation of p53tet at 0, 30 and 150 min. (B) Plot of the dissociation kinetics of p53tet with 2-exponential data fit. (C) Binding of 0.1 nM Atto 655 labeled p53tet to unlabeled p53tet. ACFs measured at unlabeled p53tet concentrations of 0, 0.05, 0.5 and 2 µM is shown. (D) Binding curve showing the M ↔ D and D ↔ T equilibrium of p53tet. Log scale is used along X-axis for clarity. The first data point was measured with sample containing only 0.1 nM of labeled p53tet.

Figure 3.

Dissociation kinetics and equilibrium constants of T-flp53 oligomerization measured using FCS. (A) Normalized ACFs following the dissociation of T-flp53 at 0, 30 and 150 min. (B) Plot of the dissociation kinetics of T-flp53 with 2-exponential fit. (C) Binding of 0.1 nM Atto 655 labeled T-flp53 to unlabeled T-flp53. ACFs measured at unlabeled T-flp53 concentrations of 0, 0.05, 0.5 and 2 µM is shown. (D) Binding curve showing the M ↔ D and D ↔ T equilibrium of flp53. Log scale is used along X-axis for clarity. The first data point was measured with sample containing only 0.1 nM of labeled flp53.

Figure 4.

DNA binding experiments of T-flp53 to Atto 655 labeled p21 and bax RE. (A) ACF of Atto 655-labeled p21 RE. Fitting to a single diffusion model yielded τ of 180 µs. (B) FCS curve of sample containing 10 nM T-flp53 and 5 nM Atto 655 labeled bax RE. No difference in diffusion time as compared to free Atto 655 labeled bax RE was observed. (C) Single component fit of the ACF obtained from the sample containing 10 nM of T-flp53 and 5 nM of Atto 655 labeled p21 RE. Residual of the fit is shown in green. (D) Two-component fit of the ACF of sample containing 10 nM of T-flp53 and 5 nM of Atto 655 labeled p21 RE (τ = 180 and 396 µs). Residual of the fit is shown in green.