Rules of helix termination by the c-capping box: calorimetric and spectroscopic studies
Thomas, Susan Tansey
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Interest in increasing the reliability of protein structural prediction has focused attention on exploring mechanisms of stabilization of helical domains by comparing the helical forming potential of each amino acid residue at different positions in the a-helix. To achieve this, detailed knowledge of thermodynamic information of protein stability with different sequences and under different solvent conditions is required and must be combined with information about the structural properties of these proteins. Using differential scanning calorimetry, circular dichroism, and fluorescence and NMR spectroscopy combined with site directed mutagenisis and chemical modifications of selected positions on the protein sequence, we studied several model systems. The ionic composition of a protein's environment is very important for its function and stability. At this time there is no common understanding of the role neutral salts play in the stabilization and destabilization of proteins. In order to address this, we studied the effects of neutral salts on the stability of the ubiquitin molecule. We found that the stability of the ubiquitin molecule increases with an increase in the concentration of electrolytes. This increase correlates with anion concentration rather than with an increase in ionic strength. This strongly suggests that the stabilizing effect occurs via specific bhiding of anions. We found an interesting interpretation of this. GdmHCl, a well known protein denaturant, can have dual actions on ubiquitin, the classical denaturant effect from the Gdm+ and a stabilizing effect from the CI. We also have complete thermodynamic analysis of more than 50 single and double, natural and non-natural amino acid substitutions at positions C-cap and C4-C" in the C-capping box of the a-helix in the ubiquitin molecule. We found that all of these variants of ubiquitin have perturbed thermodynamics of folding. From this we have generated a propensity scale of amino acid residue substitutions at the C-cap position In substitutions at positions C4 and C there were several cases in which we observed changes in the enthalpy and heat capacity of unfolding. A decrease in the enthalpy of unfolding and an increase in the heat capacity change upon unfolding is corroborated by the observation of partial cold denaturation of some of the hydrophobic variants of ubiquitin.