Folding of a three-stranded coiled coil.
Coiled coils consist of two or more amphipathic a-helices wrapped around each other to form a superhelical structure stabilized 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 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 29-residue peptide LZ16A was shown previously to be in a concentration-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 2M (k1)<==>(k(-1)) D and M + D (k2)<==>(k(-2)) T. The following rate constants were determined (25 degrees C, pH 7): k1 = 7.8 x 10(4) M(-1) s(-1), k(-1) = 0.015 s(-1), k2 = 6.5 x 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) = k1/2k(-1) and K(T) = k2/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 compatible with a two-step reaction in which a subpopulation of association competent (e.g., partly helical) monomers associate to dimeric and trimeric coiled coils.
Dürr
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The Scripps Reseach Institute, Department of Experimental Medicine & Vascular Biology MEM 275, La Jolla, Califomia 92037, USA.
Bosshard
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D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003903:Deuterium / Q000737:chemistry; D019281:Dimerization; D005470:Fluorometry / Q000379:methods; D006859:Hydrogen; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D017510:Protein Folding
D000577:Amides; D010455:Peptides; D006859:Hydrogen; D003903:Deuterium