Folding of a three–stranded coiled coil

Abstract

Coiled coils consist of two or more amphipathic α–helices wrapped around each other to form a superhelical structure stabi–lized at the interhelical interface by hydrophobic residues spaced in a repeating 3–4 sequence pattern. Dimeric coiled coils have been shown to often form in a single step reaction in which asso–ciation and folding of peptide chains are tightly coupled. Here, we ask whether such a simple folding mechanism may also apply to the formation of a three–stranded coiled coil. The designed 29–residue peptide LZ16A was shown previously to be in a concen–tration–dependent equilibrium between unfolded monomer ( M ), folded dimer ( D ), and folded trimer ( T ). We show by time–resolved fluorescence change experiments that folding of LZ16A to D and T can be described by The following rate constants were determined (25 °C, pH 7): k 1 = 7.8×10 4 M‐ 1 s‐ 1 , k‐ 1 =0.015 s‐ 1 , k 2 = 6.5×10 5 M‐ 1 s −1 , and k‐ 2 = 1.1 s‐ 1 . In a separate experiment, equilibrium binding constants were determined from the change with concentration of the far–ultraviolet circular dichroism spectrum of LZ16A and were in good agreement with the kinetic rate constants according to K D = k 1 /2k ‐1 and K T = k 2 /k‐ 2 . Furthermore, pulsed hydrogen–exchange experiments indicated that only unfolded M and folded D and T were significantly populated during folding. The results are com–patible with a two–step reaction in which a subpopulation of as–sociation competent (e.g., partly helical) monomers associate to dimeric and trimeric coiled coils.