Structure and stability of a second molten globule intermediate in the apomyoglobin folding pathway

Apomyoglobin folding proceeds through a molten globule intermediate (low- salt form; I₁) that has been characterized by equilibrium (pH 4) and kinetic (pH 6) folding experiments. Of the eight α-helices in myoglobin, three (A, G, and H) are structured in I₁, while the rest appear to be unfolded. Here we report on the structure and stability of a second intermediate, the trichloroacetate form of the molten globule intermediate (I₂), which is induced either from the acid-unfolded protein or from I₁ by ≥5 mM sodium trichloroacetate. Circular dichroism measurements monitoring urea- and acid- induced unfolding indicate that I₂ is more highly structured and more stable than I₁. Although I₂ exhibits properties closer to those of the native protein, one-dimensional NMR spectra show that it maintains the lack of fixed side-chain structure that is the hallmark of a molten globule. Amide proton exchange and ¹H-¹⁵N two-dimensional NMR experiments are used to identify the source of the extra helicity observed in I₂. The results reveal that the existing A, G, and H helices present in I₁ have become more stable in I₂ and that a fourth helix-the B helix-has been incorporated into the molten globule. Available evidence is consistent with I₂ being an on-pathway intermediate. The data support the view that apomyoglobin folds in a sequential fashion through a single pathway populated by intermediates of increasing structure and stability.