Native hydrogen bonds in a molten globule: The apoflavodoxin thermal intermediate

The structure and energetics of protein-folding intermediates are poorly un derstood. We have identified, in the thermal unfolding of the apoflavodoxin from Anabaena PCC 7119, an equilibrium intermediate with spectroscopic pro perties of a molten globule and substantial enthalpy and heat capacity of u nfolding. The structure of the intermediate is probed by mutagenesis land p hi analysis) of polar residues involved in surface-exposed hydrogen bonds c onnecting secondary-structure elements in the native protein. All hydrogen bonds analysed are formed in the molten globule intermediate, either with n ative strength or debilitated. This suggests the overall intermediate's top ology and surface tertiary interactions are close to native, and indicates that hydrogen bonding may contribute significantly to shape the conformatio n and energetics of folding intermediates. (C) 2001 Academic Press.