ata Project--Peptide Dissection

Folding and Design Laboratory

Members

John Osterhout
Sunita Kulkarni

Research

alpha-t-alpha
Peptide Dissection
HX

Group Alumni

Youcef Fezoui
Tanya Knubovets
Marcela Oslin
Diane Schaak
Wujing Xian

Protein Folding/Protein Structure Discussion Group

Talk Schedule

The alpha-t-alpha Project

Peptide Dissection

Protein Folding and Design Laboratory

The Concept

If one considers alpha-t-alpha (ata) as a peptide model for the second stage of protein folding (secondary structure association and mutual stabilization), then it is of interest to know the stability of the individual helices and the degree of their mutual stabilization. The very simple premise of this study was to synthesize peptides corresponding to the individual helices and study their stabilities separately and in the parent molecule.

Dissection of the de Novo Designed Peptide alpha-t-alpha: Stability and Properties of the Intact Molecule and Its Constituent Helices.

Fezoui, Y., Braswell, E. H., Xian, W. and Osterhout, J. J. (1999) Biochemistry 38, 2796-2804

Abstract

ata is a de novo designed 38-residue peptide (Fezoui et al, Protein Science 4: 286-295, 1995) which adopts a helical hairpin conformation in solution (Fezoui et al, (1994) Proc. Natl. Acad. Sci. USA 91, 3675-3679 and (1997) Protein Science 6: 1869-1877). Since ata was developed as a model system for protein folding at the stage where secondary structures interact and become mutually stabilizing, it is of interest to investigate the increase in stability that occurs with helix association. ata was dissected into its component helices and the relative stabilities of the individual helices and the parent molecule were assessed. The deltaG of unfolding of ata measured by guanidinium hydrochloride denaturation was determined to be 3.4 kcal/mole. The equilibrium constant for folding of ata was estimated from the deltaG as 338 and from hydrogen exchange measurements as 259. The stability of the helices in intact ata over the individual helices increased by a factor of at least 37 based on amide proton exchange measurements. Sedimentation equilibrium studies showed very little association of the peptides to form either 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 component peptides are largely reflected in the intact molecule implying that short range interactions still make important contributions to the conformation of the intact molecule even though significant stabilization is caused by helix association.