Dissection of the de novo designed peptide alpha t alpha: Stability and properties of the intact molecule and its constituent helices

alpha t alpha is a de novo designed 38-residue peptide [Fezoui et al. (1995 ) Protein Sci. 4, 286-295] that adopts a helical hairpin conformation in so lution [Fezoui et al. (1994) Proc. Natl. Acad Sci. U.S.A. 91, 3675-3679; Fe zoui et al. (1997) Protein Sci. 6, 1869-1877]. Since alpha t alpha was deve loped as a model system for protein folding at the stage where secondary st ructures interact and become mutually stabilizing, it is of interest to inv estigate the increase in stability that occurs with helix association, alph a t alpha was dissected into its component helices and the relative stabili ties of the individual helices and the parent molecule were assessed. The D elta G(0) of unfolding of alpha t alpha measured by guanidine hydrochloride denaturation was determined to be 3.4 kcal/mol. The equilibrium constant f or folding of alpha t alpha: was estimated from the Delta G(0) as 338 and f rom hydrogen exchange measurements as 259. The stability of the helices in intact alpha t alpha over the individual helices increased by a factor of a t least 37 based on amide proton exchange measurements. Sedimentation equil ibrium studies showed very little association of the peptides to form eithe r homo- or heterodimers, suggesting that helix association is stabilized by the high effective concentration of the helices caused by the presence of the connecting turn. The effects of salt and pH on the helicity of the comp onent peptides are largely reflected in the intact molecule, implying that short-range interactions still make important contributions to the conforma tion of the intact molecule even though significant stabilization is caused by helix association.