Distance measurements in the borderline region of applicability of CW EPR and DEER: A model study on a homologous series of spin-labelled peptides

Inter-spin distances between 1nm and 4.5nm are measured by continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) methods for a series of nitroxide-spin-labelled peptides. The upper distance limit for measuring dipolar coupling by the broadening of the CW spectrum and the lower distance limit for the present optimally-adjusted double electron electron resonance (DEER) set-up are determined and found to be both around 1.6–1.9nm. The methods for determining distances and corresponding distributions from CW spectral line broadening are reviewed and further developed. Also, the work shows that a correction factor is required for the analysis of inter-spin distances below approximately 2nm for DEER measurements and this is calculated using the density matrix formalism.