Many oncogenic mutations inactivate the tumor suppressor p53 by destabilizing it, leading to its rapid aggregation. Small molecule drugs are being developed to stabilize such mutants. The kinetics of aggregation of p53 is deceptively simple. The initial steps in the micromolar concentration range follow apparent sigmoidal sequential first-order kinetics, with rate constants k 1 and k 2 . However, the aggregation kinetics of a panel of mutants prepared for Φ-value analysis has now revealed a bimolecular reaction hidden beneath the observed first-order kinetics. Φ u measures the degree of local unfolding on a scale of 0-1. A number of sequential Φ uvalues of ∼1 for k 1 and k 2 over the molecule implied more than one protein molecule must be reacting, which was confirmed by finding a clear concentration dependence at submicromolar protein. Numerical simulations showed that the kinetics of the more complex mechanism is difficult, if not impossible, to distinguish experimentally from simple first order under many reaction conditions. Stabilization of mutants by small molecules will be enhanced because they decrease both k 1 and k 2 . The regions with high Φ u -values point to the areas where stabilization of mutant proteins would have the greatest effect.protein | amyloid | folding | misfolding | cancer T he p53 tumor suppressor plays an essential role in genome surveillance and protects against cancer (1-5). p53 is inactivated in more than 70% of cancer types by missense point mutations, mainly located in the DNA-binding core domain (6-8). WT p53, with a melting temperature of 45°C (9), is intrinsically thermodynamically unstable (10), as well as kinetically (11). Many of the oncogenic mutations further destabilize p53, leading to its rapid aggregation at body temperature (10-13). The increased aggregation propensity of the destabilizing mutants, such as R175H, Y220C, and R249S, not only causes loss of function but is also suggested to exert a dominant negative function by coaggregation with WT p53 (12, 14), p63, and p73 (15-19). p53 mutants with higher aggregation propensity are reported to impair p73-and p63-mediated transcription and apoptosis (15). These p53 mutants accumulate at high levels in most of tumors due to resistance to proteasome-dependent degradation. The high expression of mutant p53 in tumors makes stabilizing the mutants and restoring their WT functions an attractive strategy to eliminate selectively tumor cells.Small molecules have been developed to stabilize such destabilizing mutants (20). Many of them are from cell or in vitro screening (21, 22), whereas others are rationally designed from the structures of WT p53 and mutants (21, 23). These small molecules can effectively restore WT activity of mutant p53 and specifically kill cancer cells.The best-studied examples of protein aggregation are those that form regular repeating fibrillar structures (24-27). p53 aggregates have some of the diagnostic features of such fibrils, such as the binding of the dye thioflavine T (ThT), and will form regu...