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</i>, <i>i+</i>3), (<i>i</i>, <i>i+</i>4), and (<i>i</i>, <i>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</i>, <i>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</i>, <i>i+</i>3) interactions, whereas any Xaa side chain length supported Zbb-Xaa (<i>i</i>, <i>i+</i>4) interactions. In particular, the magnitude of both Zbb^–-Lys (<i>i</i>, <i>i+</i>4) and Zbb^–-Orn (<i>i</i>, <i>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</i>, <i>i+</i>3) interactions involved the χ₁ dihedral combination (<i>g</i>+, <i>g</i>+) for the <i>i</i> and <i>i</i>+3 residues, whereas the Zbb-Xaa (<i>i</i>, <i>i+</i>4) interactions were supported by the χ₁ dihedral combination (<i>t</i>, <i>g</i>+) for the <i>i</i> and <i>i</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

Effect of Charged Amino Acid Side Chain Length at Non-Hydrogen Bonded Strand Positions on β‑Hairpin Stability

β-Sheets have been implicated in various neurological disorders, and ∼20% of protein residues adopt a sheet conformation. Therefore, studies on the structural origin of sheet stability can provide fundamental knowledge with potential biomedical applications. Oppositely charged amino acids are frequently observed across one another in antiparallel β-sheets. Interestingly, the side chains of natural charged amino acids Asp, Glu, Arg, Lys have different numbers of hydrophobic methylenes linking the backbone to the hydrophilic charged functionalities. To explore the inherent effect of charged amino acid side chain length on antiparallel sheets, the stability of a designed hairpin motif containing charged amino acids with varying side chain lengths at non-hydrogen bonded positions was studied. Peptides with the guest position on the N-terminal strand and the C-terminal strand were investigated by NMR methods. The charged amino acids (Xaa) included negatively charged residues with a carboxylate group (Asp, Glu, Aad in increasing length), positively charged residues with an ammonium group (Dap, Dab, Orn, Lys in increasing length), and positively charged residues with a guanidinium group (Agp, Agb, Arg, Agh in increasing length). The fraction folded and folding free energy for each peptide were derived from the chemical shift deviation data. The stability of the peptides with the charged residues at the N-terminal guest position followed the trends: Asp > Glu > Aad, Dap < Dab < Orn ∼ Lys, and Agb < Arg < Agh < Agp. The stability of the peptides with the charged residues at the C-terminal guest position followed the trends: Asp < Glu < Aad, Dap ∼ Dab < Orn ∼ Lys, and Agb < Arg ∼ Agp < Agh. These trends were rationalized by thermodynamic sheet propensity and cross-strand interactions