Energetics of coiled coil folding: the nature of the transition states.

Abstract

Coiled coils are simple models for studying the association of two polypeptide chains to form a folded protein. Previous work has shown that the folding of a coiled coil can be described by a two-state transition between two unfolded monomeric peptide chains and a folded coiled coil dimer. Here we report the thermodynamic activation parameters for the folding and unfolding of two unrelated coiled coils: C62GCN4 and A(2). C62GCN4 corresponds to the 62 C-terminal residues of yeast transcription factor GCN4. The peptide forms a dimeric coiled coil through its 33 C-terminal residues. A(2) is a designed 30-residue dimeric coiled coil whose folding is induced by low pH [Dürr, E., Jelesarov, I., and Bosshard, H. R. (1999) Biochemistry 38, 870-880]. Folding and unfolding were assessed under identical native buffer conditions so that the microscopic reversibility applied and the transition state was the same for folding and unfolding. The time course of folding was followed from the self-quenching of a C-terminal fluorescent label (Texas Red). The overall folding of both peptides is enthalpy-driven and opposed by a loss of entropy. The main energetic changes occur after the system has passed the transition state. In the folding of C62GCN4, only 10-20% of the heat capacity change is attained between the monomeric state and the dimeric transition state. For coiled coil A(2), the fractional heat capacity change preceding the transition state is 30-40%. The results indicate that the activated states of folding of coiled coils are not well structured and differ considerably from the folded coiled coil conformation. These findings are in agreement with a rate-limiting transition state in which the coiled coil helices and the hydrophobic coiled coil interface are poorly developed.