Contribution of the 30/36 hydrophobic contact at the C-terminus of the α-helix to the stability of the ubiquitin molecule

The contribution of the hydrophobic contact in the C-capping motif of the alpha-helix to the thermodynamic stability of the ubiquitin molecule has been analyzed. For this, 16 variants of ubiquitin containing the full combinatorial set of four nonpolar residues Val, Ile, Leu, and Phe at C4 (Ile30) and C " (Ile36) positions were generated. The secondary structure content as estimated using far-UV circular dichroism (CD) spectroscopy of all but Phe variants at position 30 did not show notable changes upon substitutions. The thermodynamic stability of these ubiquitin variants was measured using differential scanning calorimetry, and it was shown that all variants have lower stability as measured by decreases in the Gibbs energy. Since in some cases the decrease in stability was so dramatic that it rendered an unfolded protein, it was therefore concluded that, despite apparent preservation of the secondary structure, the 30/36 hydrophobic contact is essential for the stability of the ubiquitin molecule. The decrease in the Gibbs energy in many cases was found to be accompanied by a large (up to 25%) decrease in the enthalpy of unfolding, particularly significant in the Variants containing Ile to Leu substitutions. This decrease in enthalpy of unfolding is proposed to be primarily the result of the perturbed packing interactions in the native state of the lie --> Leu variants. The analysis of these data and comparison with effects of similar amino acid substitutions on the stability of other model systems suggest that ne --> Leu substitutions cannot be isoenergetic at the buried site.