Electron spin resonance and structural analysis of water soluble, alanine-rich peptides incorporating TOAC

The unnatural, conformationally constrained nitroxide amino acid TOAC, a C-alpha,C- alpha-disubstituted glycine, stabilizes helical structure and provides a means for studying rigidly spin labelled peptides by electron spin resonance. Water soluble, alanine-rich sixteen and twenty residue sequences are examined with single and double TOAC labelling. Lineshape analysis of the singly labelled peptide reveals evidence of substantial anisotropic rotational diffusion. Combined circular dichroism and electron spin resonance of the double labelled sequences demonstrates that both the 16-residue and 20-residue peptides are completely folded and adopt predominantly the alpha-helical conformation. However, lineshape fitting of the 16-residue sequences cooled in MeOH revealed a surprising result. Instead of the typical alpha-helix geometry of 3.6 residues per turn found in peptide and protein crystal structures, these doubly TOAC labelled peptides adopt a more open geometry of approximately 3.8-3.9 residues per turn. Although this conformation shift is subtle, it nevertheless suggests that helical peptides in solution are plastic structures that may readily adopt a wide range of conformations depending on context and local solvation.