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Intrinsically disordered p53 transactivation domain and its helically pre-structured segment. — The strengths of the water-protein interactions for intrinsically disordered proteins (IDPs) vary in a narrow range, and are nearer to the bulk-phase water-water interactions than to that for globular proteins (GPs, e.g. bovine serum albumin, BSA). The “surface” of IDPs is more open to the water, and the amount of the bound water molecules involved is much greater than for GPs. Wide-line 1H NMR intensity and differential scanning calorimetry (DSC) measurements were carried out on the intrinsically disordered 73-residue full transactivation domain (TAD) of the p53 tumor suppressor protein and two peptides: one a wild type p53 TAD peptide with a helix pre-structuring property, and a mutant peptide with a disabled helix-forming propensity. The number of the interacting protein-water connections were quantified. The wild-type p53 TAD has a highly disordered structure as compared to BSA, a reference GP. We found the degree of disorder (being less structured) to increase as BSA < wild type full p53 TAD < wild type p53 TAD helix peptide < mutant p53 TAD peptide. The DSC measurements show the interactions of the proteins with the other solutes, especially with sodium and chloride ions. The full p53 TAD and the peptides bind a considerably greater amount of NaCl than the globular BSA. Both methods, wide-line 1H-NMR and DSC, are not only capable of distinguishing IDPs against GPs, but are also able to differentiate two structural states of IDPs in terms of their interactions with water molecules or other chemical entities, such as salt ions (Fig. 1).

Figure 1. Left panel: Hydration measured by the 1H-NMR signal intensities of the mobile water for 50 mg/ml p53 TAD (blue) and 50 mg/ml BSA (black) dissolved in water. T = −4 °C data for the helices are shown for comparison (green and red). Right panel: Enthalpy changes associated with the eutectic melting of the H2O-NaCl system measured as a function of protein concentration for full p53 TAD (blue), wild type p53 TAD helix peptide (green) and mutant peptide (red) dissolved in the buffer solution of 150 mM NaCl, 50 mM Tris, 1 mM EDTA, pH = 7.5. The lines are guides to the eye.