Thermodynamic analysis of a designed three-stranded coiled coil.
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, delta Cp, 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 delta Cp of 750 cal deg-1 mol-1. This translates to a delta Cp 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.
Boice
J A
JA
Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000, USA.
Dieckmann
G R
GR
DeGrado
W F
WF
Fairman
R
R
D002942:Circular Dichroism; D008956:Models, Chemical; D008958:Models, Molecular; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012984:Software; D013816:Thermodynamics