Unfolding of alanine-based peptides using electron spin resonance distance measurements.

We describe a scheme for tagging an alanine-based peptide with a Cu(II) and a nitroxide to measure unfolding transitions. The enhancement in longitudinal relaxation rate of the nitroxide due to the presence of Cu(II) was measured at physiological temperatures by pulsed electron spin resonance (ESR). The change in relaxation rate provided the average interspin distance between the Cu(II) and the nitroxide. Control experiments on a proline-based peptide verify the robustness of the method. The change in interspin distances with temperature for the alanine-based peptide is in accord with the change in helicity measured by circular dichroism. The data provide an opportunity to examine the unfolding process in polyalanine peptides. The distance in the folded state is in concordance with molecular dynamics. However, the ESR experiment measures an average distance of 17 A in the unfolded state, whereas molecular dynamics indicates a distance of 42 A if the unfolded geometry was a polyproline type II helix. Therefore, ESR demonstrates that the unfolded state of this alanine-based peptide is not an ideal extended polyproline type II helix.