The preferred conformation of the tripeptide Ala-Phe-Ala in water is an inverse γ-turn: Implications for protein folding and drug design

Recent studies have provided evidence that peptides as short as tripeptides do adopt preferred conformations. Here we report that the tripeptide Ala-Phe-Ala (AFA) in aqueous solution preferentially forms an inverse γ-turn. Circular dichroism (CD) indicated the presence of a predominant turn structure, and Fourier transform infrared (FTIR) bands suggested the presence of a γ-turn forming a bifurcated H-bond with the solvent molecules. The high-resolution structure was obtained by a combined use of NMR spectroscopy and calculations. On the basis of 30 unambiguous ROESY-derived distance restraints (including the Hα-NH NOE between Ala¹ and Ala³ and a hydrogen bond between the CO group of Ala¹ and the NH group of Ala³), calculations clearly demonstrated the presence of an inverse γ-turn centered on Phe². From NOE data, we estimated a mole fraction for the γ-turn of 0.65. Since for AFA an extended β-strand was also reported [Eker, F., Griebenow, K., Cao, X., Nafie, L. A., and Schweitzer-Stenner, R. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 10054-10059], we investigated the possibility that γ-turn and β-strand may represent two major conformations. By using a best-fit procedure that calculated experimental NOEs as weighted averages of the effects originating from both structures, we were able to calculate with good accuracy the backbone NOEs at 280 K in terms of the two limiting conformers, yielding a mole fraction for the γ-turn and β-strand conformations of 0.60 and 0.40, respectively, in good agreement with those found by NOE data. The implication of the existence of a preferred conformation by a small structural element is discussed in the context of the nucleation of protein folding events and the design of small peptide and peptidomimetic drugs.