Formation of a molten-globule-like state of myoglobin in aqueous hexafluoroisopropanol.

The effects of aqueous hexafluoroisopropanol (HFIP) media on the structure of myoglobin are reported. Circular dichroism (CD) spectra of this alpha-helical protein in as little as 4% (v/v) HFIP indicate that native-like amounts of secondary structure remain while rigid tertiary structure is lost. However, thermal studies suggest some residual cooperativity of unfolding in this state. At much higher HFIP concentrations, the helicity exceeds the native value and the protein behaves as a series of independent helices which do not interact with each other. We did not observe cold denaturation of myoglobin, even though this phenomenon has been observed for molten globule states of myoglobin, as well as for monomeric amphipathic alpha-helices when moderate quantities of HFIP are present. The pH dependence of trifluoroethanol-induced disruption of tertiary structure revealed that the degree of disruption increases as the enthalpic advantage of the folded state is diminished at low pH.