Fluoroalcohols as structure modifiers in peptides and proteins: hexafluoroacetone hydrate stabilizes a helical conformation of melittin at low pH

The effect of hexafluoroacetone hydrate (HFA) on the structure of the honey bee venom peptide melittin has been investigated. In aqueous solution at l ow pH melittin is predominantly unstructured. Addition of HFA at pH approxi mate to 2.0 induces a structural transition from the unstructured state to a predominantly helical conformation as suggested by intense diagnostic far UV CD bands. The structural transition is highly cooperative and complete at 3.6 M (50% v/v) HFA. A similar structural transition is also observed in 2,2,2 trifluoroethanol which is complete only at a cosolvent concentration of approximate to 8 M. Temperature dependent CD experiments support a 'col d denaturation' of melittin at low concentrations of HFA, suggesting that s elective solvation of peptide by HFA is mediated by hydrophobic interaction s. NMR studies in 3.6 M HFA establish a well-defined helical structure of m elittin at low pH, as suggested by the presence of strong NHi/NHi+1,I NOEs throughout the sequence, along with many medium range helical NOEs. Structu re calculations using NOE-driven distance constraints reveal a well-ordered helical fold with a relatively flexible segment around residues T10-G11-T1 2. The helical structure of melittin obtained at 3.6 M HFA at low pH is sim ilar to those determined in methanolic solution and perdeuterated dodecylph osphocholine micelles. HFA as a cosolvent facilitates helix formation even in the highly charged C-terminal segment.