Peptide destabilization by two adjacent D-amino acids in single-stranded amphipathic alpha-helices

Area of Science:

• Peptide chemistry and structural biology.
• Biophysical characterization of protein and peptide structure.
• Amino acid chemistry and its impact on peptide conformation.

Background:

• Amphipathic alpha-helices are crucial structural motifs in peptides.
• D-amino acid substitutions can alter peptide stability and function.
• Previous work identified local destabilizing effects of D-amino acid replacements.

Purpose of the Study:

• To quantify the destabilizing effect of two adjacent D-amino acids in the center of an amphipathic alpha-helix.
• To investigate the impact of these substitutions on secondary structure stability.
• To confirm the contribution of helical stability energy to peptide behavior in solution.

Main Methods:

• Hydrogen exchange studies to monitor backbone proton dynamics.
• Guanidine hydrochloride denaturation experiments to assess structural stability.
• Spectroscopic analysis in trifluoroethanol (TFE)/water solvent mixtures.

Main Results:

• Double D-amino acid incorporation at the helix center destabilized secondary structure by 4.5 kJ/mol.
• A significant (10-fold) variation in exchange rate was observed for one backbone proton.
• Other backbone protons showed minimal influence from the D-amino acid substitution.

Conclusions:

• The energy contribution of -4.5 kJ/mol per residue is significant for helical peptide stability.
• Double D-amino acid replacements provide a tool for characterizing helical peptides.
• Results validate the hypothesis regarding the energetic contribution to helical stability in TFE/water mixtures.