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 prov ides a means for studying rigidly spin labelled peptides by electron spin r esonance. Water soluble, alanine-rich sixteen and twenty residue sequences are examined with single and double TOAC labelling. Lineshape analysis of t he singly labelled peptide reveals evidence of substantial anisotropic rota tional diffusion. Combined circular dichroism and electron spin resonance o f the double labelled sequences demonstrates that both the 16-residue and 2 0-residue peptides are completely folded and adopt predominantly the alpha- helical conformation. However, lineshape fitting of the 16-residue sequence s 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 cryst al structures, these doubly TOAC labelled peptides adopt a more open geomet ry of approximately 3.8-3.9 residues per turn. Although this conformation s hift 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.