Mutations in p53's DNA binding domain (p53DBD) are associated with 50% of all cancers, making it an essential system to investigate and understand the genesis and progression of cancer. In this work, we studied the changes in the structure and dynamics of wild type p53DBD in comparison with two of its "hot-spot" DNA-contact mutants, R248Q and R273H, by analysis of backbone amide chemical shift perturbations and N spin relaxation measurements. The results of amide chemical shift changes indicated significantly more perturbations in the R273H mutant than in wild type and R248Q p53DBD. Analysis ofN spin relaxation rates and the resulting nuclear magnetic resonance order parameters suggests that for most parts, the R248Q mutant exhibits limited conformational flexibility and is similar to the wild type protein. In contrast, R273H showed significant backbone dynamics extending up to its β-sandwich scaffold in addition to motions along the DNA binding interface. Furthermore, comparison of rotational correlation times between the mutants suggests that the R273H mutant, with a higher correlation time, forms an enlarged structural fold in comparison to the R248Q mutant and wild type p53DBD. Finally, we identify three regions in these proteins that show conformational flexibility to varying degrees, which suggests that the R273H mutant, in addition to being a DNA-contact mutation, exhibits properties of a conformational mutant.

The present study explored both spontaneous and stress-induced deamidation in acid trehalase and endo-xylanase. An alteration in optimum pH by 1.5 units and optimum temperature by 20 °C accelerated the process of deamidation with a rise in isoaspartate formation and ammonia loss. Spontaneous deamidation during an enzyme-substrate reaction at physiological conditions resulted in accretion of isoaspartyl residues within the enzymes which gradually impaired their catalytic efficacy. Deamidation appeared to be more pronounced in endo-xylanase owing to its secondary structure conformation and high asparagine content. The active sites, Ala 549 in acid trehalase and His184 and Trp188 in endo-xylanase contributed to the loss of enzyme activity as they were flanking the deamidation-susceptible Asn residues. Protein L-isoaspartyl methyl transferase seemed to have a repairing capability, which enabled the heat-damaged enzymes to regain their partial activity as evident from there rise in K (cat)/K (m). Endo-xylanase could regain 38.1 % of its biological activity while a lesser 17.5 % reactivation was obtained in acid trehalase. A unique protein L-isoaspartyl methyl transferase recognition site, Asn 151 was also identified in acid trehalase. A mass increment of the tryptic peptides of repaired enzyme due to methylation catalyzed by protein L-isoaspartyl methyl transferase substantiated the repair hypothesis.

Mutations in the core domain of tumor suppressor protein p53 have been associated with ∼50% of the occurrences of human cancers. A majority of these mutations inactivate p53 function by destabilizing its native structure. Although studies have shown p53’s function can be restored by stabilizing the mutants to their wild-type conformation with immense therapeutic potential, its applicability has been restricted because of our limited understanding of the precise nature of destabilization arising from changes in the mutant p53’s structure and dynamics. Here, using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations, we have probed the conformational flexibility in three of the most widespread and clinically important “hot spot” mutants of the p53 core domain. Our results show that NMR order parameter-derived conformational entropy is linearly correlated with the change in free energy of urea-mediated denaturation, the latter being a well-established reporter of stability in p53 core domain mutants. Using a linear regression function, we show that the three parameters of equilibrium denaturation experiments, i.e., the free energy of denaturation (ΔG D–N H2O), the slope of the transition (m), and the urea concentration at 50% denaturation ([urea]50%), can be used to predict the conformational entropy in p53 core domain mutants, thereby demonstrating a method for using these parameters as predictors of a protein’s conformational entropy, which has been known to shape the functional properties of proteins.

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