Thermodynamic Analysis of a Designed Three-Stranded Coiled Coil

Abstract

The study and successful design of coiled-coil protein structural motifs have provided much insight into the rules governing protein folding and stability. In this work we use a thermodynamic approach to quantitate the rules that govern the specific oligomerization of coiled coils. We have designed a highly stable trimeric coiled coil by placing valine residues at each a position and leucine residues at each d position of the heptad repeating unit. The peptide forms a very stable trimer as determined by sedimentation equilibrium, and the concentration dependence of its circular dichroism spectrum follows a cooperative monomer/dimer/trimer equilibrium with the dimer state as a highly unstable intermediate. Its guanidinium chloride denaturation curve was collected at several peptide concentrations, and analysis of the data confirms the cooperativity of the trimerization process and provides a free energy of stabilization of −18.4 kcal mol^-1 for the trimer. The heat capacity, ΔC_p, was measured by global analysis of thermal unfolding data collected at a number of guanidinium chloride concentrations. Guanidinium chloride induces cold denaturation in the thermal unfolding curves, providing a reasonably well-determined value for ΔC_p of 750 cal deg^-1 mol^-1. This translates to a ΔC_p of 8.6 cal deg^-1 mol^-1 per residue and corresponds well to that expected of a coiled coil with a well-defined tertiary structure.