Understanding the Folding Mechanism of an -Helical Hairpin
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-helical hairpin is the fundamental building block of the widespread helix-turn-helixDNA binding motif. With two antiparallel helices connected by a reverse turn, the -helical hairpin structuremay be regarded as a "supersecondary structural element" and, therefore, could exhibit rather uniquefolding properties. So far, the folding mechanism of -helical hairpins has not been studied in detail andremains elusive. Herein, we examine the effects of the turn, the hydrophobic cluster, and a disulfidecross-linker on the folding kinetics of a designed -helical hairpin, Z34C, using an infrared temperature-jump (T-jump) method in conjunction with site-specific mutagenesis. Our results show that Z34C foldswith an ultrafast rate (~4.0 × 105 s-1) and support a folding mechanism in which the rate-limiting stepcorresponds to the formation of the reverse turn. On the other hand, the hydrophobic cluster and thedisulfide cross-linker appear to largely stabilize the native state but not the folding transition state.
-helical hairpin is the fundamental building block of the widespread helix-turn-helixDNA binding motif. With two antiparallel helices connected by a reverse turn, the -helical hairpin structuremay be regarded as a "supersecondary structural element" and, therefore, could exhibit rather uniquefolding properties. So far, the folding mechanism of -helical hairpins has not been studied in detail andremains elusive. Herein, we examine the effects of the turn, the hydrophobic cluster, and a disulfidecross-linker on the folding kinetics of a designed -helical hairpin, Z34C, using an infrared temperature-jump (T-jump) method in conjunction with site-specific mutagenesis. Our results show that Z34C foldswith an ultrafast rate (~4.0 × 105 s-1) and support a folding mechanism in which the rate-limiting stepcorresponds to the formation of the reverse turn. On the other hand, the hydrophobic cluster and thedisulfide cross-linker appear to largely stabilize the native state but not the folding transition state.