Effect of glutamate side chain length on intrahelical glutamate-lysine ion pairing interactions.

Ion pairing interactions between oppositely charged amino acids are important for protein structure stability. Despite the apparent electrostatic nature of these interactions, the charged amino acids Lys, Arg, Glu, and Asp have a different number of hydrophobic methylenes linking the charged functionality to the backbone. To investigate the effect of Glu (and Asp) side chain length on ion pairing interactions, a series of 36 monomeric α-helical peptides containing Zbb-Xaa (i, i+3), (i, i+4), and (i, i+5) (Zbb = Aad, Glu, Asp; Xaa = Lys, Orn, Dab, Dap) sequence patterns were studied by circular dichroism (CD) spectroscopy at pH 7 and 2. Peptides with Glu and Aad exhibited similar helicity and pH dependence, whereas peptides with Asp behaved distinctly different. The side chain interaction energetics were derived from the CD data using the nesting block method coupled with modified Lifson-Roig theory. At pH 7, no Zbb-Xaa (i, i+5) interaction was observed, regardless of side chain length (consistent with the helix geometry). Interestingly, only Lys was capable of supporting Zbb-Xaa (i, i+3) interactions, whereas any Xaa side chain length supported Zbb-Xaa (i, i+4) interactions. In particular, the magnitude of both Zbb(-)-Lys (i, i+4) and Zbb(-)-Orn (i, i+4) interaction energies followed the trend Asp > Glu > Aad. Side chain conformational analysis by molecular mechanics calculations showed that the Zbb-Xaa (i, i+3) interactions involved the χ(1) dihedral combination (g+, g+) for the i and i+3 residues, whereas the Zbb-Xaa (i, i+4) interactions were supported by the χ(1) dihedral combination (t, g+) for the i and i+4 residues. These calculated low energy conformers were consistent with conformations of intrahelical Asp-Lys and Glu-Lys salt bridges in a nonredundant protein structure database. These results suggest that Asp and Glu provide natural variation, and lengthening the Glu side chain further to Aad does not furnish additional characteristics that Glu cannot supply.

Positional effects on helical Ala-based peptides.

Helix-coil equilibrium studies are important for understanding helix formation in protein folding, and for helical foldamer design. The quantitative description of a helix using statistical mechanical models is based on experimentally derived helix propensities and the assumption that helix propensity is position-independent. To investigate this assumption, we studied a series of 19-residue Ala-based peptides, to measure the helix propensity for Leu, Phe, and Pff at positions 6, 11, and 16. Circular dichroism spectroscopy revealed that substituting Ala with a given amino acid (Leu, Phe, or Pff) resulted in the following fraction helix trend: KXaa16 > KXaa6 > KXaa11. Helix propensities for Leu, Phe, and Pff at the different positions were derived from the CD data. For the same amino acid, helix propensities were similar at positions 6 and 11, but much higher at position 16 (close to the C-terminus). A survey of protein helices revealed that Leu/Phe-Lys (i, i + 3) sequence patterns frequently occur in two structural patterns involving the helix C-terminus; however, these cases include a left-handed conformation residue. Furthermore, no Leu/Phe-Lys interaction was found except for the Lys-Phe cation-π interaction in two cases of Phe-Ala-Ala-Lys. The apparent high helix propensity at position 16 may be due to helix capping, adoption of a 310-helix near the C-terminus perhaps with Xaa-Lys (i, i + 3) interactions, or proximity to the peptide chain terminus. Accordingly, helix propensity is generally position-independent except in the presence of alternative structures or in the proximity of either chain terminus. These results should facilitate the design of helical peptides, proteins, and foldamers.