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Articles by A. R. Fersht
Total Records ( 2 ) for A. R. Fersht
  K. H Khoo , A. C Joerger , S. M.V Freund and A. R. Fersht
 

The core domain of the tumour suppressor p53 is of inherently low thermodynamic stability and also low kinetic stability, which leads to rapid irreversible denaturation. Some oncogenic mutations of p53 act by just making the core domain thermosensitive, and so it is the target of novel anti-cancer drugs that bind to and stabilise the protein. Increasing the stability of the unstable core domain has also been crucial for biophysical and structural studies, in which a stabilised quadruple mutant (QM) is currently used. We generated an even more stabilised hexamutant (HM) by making two additional substitutions, Y236F and T253I, to the QM. The residues are found in the more stable paralogs p63 and p73 and stabilise the wild-type p53 core domain. We solved the structure of the HM core domain by X-ray crystallography at 1.75 Å resolution. It has minimal structural changes from QM that affect the packing of hydrophobic core residues of the β-sandwich. The full-length HM was also fully functional in DNA binding. HM was more stable than QM at 37°C. Anomalies in biophysics and spectroscopy in urea-mediated denaturation curves of HM implied the accumulation of a folding intermediate, which may be related to those detected in kinetic experiments. The two additional mutations over-stabilise an unfolding intermediate. These results should be taken into consideration in drug design strategies for increasing the stability of temperature-sensitive mutants of p53.

  C. A Dodson , N Ferguson , T. J Rutherford , C. M Johnson and A. R. Fersht
 

The SAP domain from the Saccharomyces cerevisiae THO1 protein contains a hydrophobic core and just two -helices. It could provide a system for studying protein folding that bridges the gap between studies on isolated helices and those on larger protein domains. We have engineered the SAP domain for protein folding studies by inserting a tryptophan residue into the hydrophobic core (L31W) and solved its structure. The helical regions had a backbone root mean-squared deviation of 0.9 Å from those of wild type. The mutation L31W destabilised wild type by 0.8 ± 0.1 kcal mol–1. The mutant folded in a reversible, apparent two-state manner with a microscopic folding rate constant of around 3700 s–1 and is suitable for extended studies of folding.

 
 
 
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