The reversible folding and unfolding of barley chymotrypsin inhibitor 2 (CI2) appears to be a rare example in which both equilibria and kinetics are described by a two-state model. Equilibrium denaturation by guanidinium chloride and heat is completely reversible, and the data can be fitted to a simple two-state model involving only native and denatured forms. The free energy of folding in the absence of denaturant, delta GH2O, at pH 6.3, is calculated to be 7.03 +/- 0.16 and 7.18 +/- 0.43 kcal mol-1 for guanidinium chloride and thermal denaturation, respectively. Scanning microcalorimetry shows that the ratio of the van't Hoff enthalpy of denaturation to the calorimetric enthalpy of denaturation does not deviate from unity, the value observed for a two-state transition, over the pH range 2.2-3.5. The heat capacity change for denaturation is found to be 0.789 kcal mol-1 K-1. The rate of unfolding of CI2 is first order and increases exponentially with increasing guanidinium chloride concentration. Refolding, however, is complex and involves at least three well-resolved phases. The three phases result from heterogeneity of the unfolded form due to proline isomerization. The fast phase, 77% of the amplitude, corresponds to the refolding of the fraction of the protein that has all its prolines in a native trans conformation. The rate of this major phase decreases exponentially with increasing guanidinium chloride concentration. The unfolding and refolding kinetics can also be fitted to a two-state model.(ABSTRACT TRUNCATED AT 250 WORDS)

Currently, around 11 million people are living with a tumour that contains an inactivating mutation of TP53 (the human gene that encodes p53) and another 11 million have tumours in which the p53 pathway is partially abrogated through the inactivation of other signalling or effector components. The p53 pathway is therefore a prime target for new cancer drug development, and several original approaches to drug discovery that could have wide applications to drug development are being used. In one approach, molecules that activate p53 by blocking protein-protein interactions with MDM2 are in early clinical development. Remarkable progress has also been made in the development of p53-binding molecules that can rescue the function of certain p53 mutants. Finally, cell-based assays are being used to discover compounds that exploit the p53 pathway by either seeking targets and compounds that show synthetic lethality with TP53 mutations or by looking for non-genotoxic activators of the p53 response.

The role of complementary hydrogen bonding as a determinant of biological specificity has been examined by protein engineering of the tyrosyl-tRNA synthetase. Deletion of a side chain between enzyme and substrate to leave an unpaired, uncharged hydrogen-bond donor or acceptor weakens binding energy by only 0.5-1.5 kcal mol-1. But the presence of an unpaired and charged donor or acceptor weakens binding by a further approximately 3 kcal mol-1.

In the transition state for unfolding of barnase, the hydrophobic core between the major alpha-helix and beta-sheet is somewhat weakened, the C terminus of the major helix is largely intact but its N terminus is exposed and a major loop has been invaded by solvent.

The rapid association of barnase and its intracellular inhibitor barstar has been analysed from the effects of mutagenesis and electrostatic screening. A basal association rate constant of 10(5) M(-1) s(-1) is increased to over 5 x 10(9) M(-1) s(-1) by electrostatic forces. The association between the oppositely charged proteins proceeds through the rate-determining formation of an early, weakly specific complex, which is dominated by long-range electrostatic interactions, followed by precise docking to form the high affinity complex. This mode of binding is likely to be used widely in nature to increase association rate constants between molecules and its principles may be used for protein design.

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The tumor suppressor protein p53 induces or represses the expression of a variety of target genes involved in cell cycle control, senescence, and apoptosis in response to oncogenic or other cellular stress signals. It exerts its function as guardian of the genome through an intricate interplay of independently folded and intrinsically disordered functional domains. In this review, we provide insights into the structural complexity of p53, the molecular mechanisms of its inactivation in cancer, and therapeutic strategies for the pharmacological rescue of p53 function in tumors. p53 emerges as a paradigm for a more general understanding of the structural organization of modular proteins and the effects of disease-causing mutations.