Folding of a three-stranded coiled coil

Coiled coils consist of two or more amphipathic cu-helices wrapped around e ach other to form a superhelical structure stabilized at the interhelical i nterface by hydrophobic residues spaced in a repeating 3-4 sequence pattern . Dimeric coiled coils have been shown to often form in a single step react ion in which association 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 29residue peptide LZ16A was shown previously to be in a concentration-dependent equilibrium b etween unfolded monomer (M), folded dimer (D), and folded trimer (T). We sh ow by time-resolved fluorescence change experiments that folding of LZ16A t o D and T can be described by 2M reversible arrow(k-1)(k1) D and M + D reversible arrow(k-2)(k2) T. The following rate constants were determined (25 degrees C, pH 7): k(1) 7.8 x 10(4) M-1 s(-1), k(-1), = 0.015 s(-1), kz = 6.5 x 10(5) M-1 s(-1), and k (-2) = 1.1 s(-1). In a separate experiment, equilibrium binding constants w ere determined from the change with concentration of the far-ultraviolet ci rcular dichroism spectrum of LZ16A and were in good agreement with the kine tic rate constants according to K-D = k(1)/2k(-1) and K-T = k(2)/k(-2) Furt hermore, pulsed hydrogen-exchange experiments indicated that only unfolded M and folded D and T were significantly populated during folding. The resul ts are compatible with a two-step reaction in which a subpopulation of asso ciation competent (e.g., partly helical) monomers associate to dimeric and trimeric coiled coils.