Cold-denatured ensemble of apomyoglobin: Implications for the early steps of folding

The dynamics of protein-refolding experiments initiated by a temperature jump depend critically on the nature of the initial cold-denatured ensemble. The cold-denatured state of equine apomyoglobin has been investigated in aqueous buffers by near- and far-UV circular dichroism, fluorescence, infrared, and NMR spectroscopies at temperatures ranging from -20 to 98 °C. Cold denaturation of apomyoglobin is well described by a cooperative transition below 3 °C and differs in many aspects from acid-induced unfolding. As a reference system, the N-terminal A-peptide fragment of equine apomyoglobin has also been studied in aqueous and trifluoroethanol solutions. The A-peptide has a low helix-forming propensity in the absence of any stabilizing tertiary interactions. The results show that cold denaturation breaks the AGH-hydrophobic interface of equine apomyoglobin. Furthermore, at least some GH-helical structure appears to be preserved at the expense of the less stable A-helix.