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Inter-spin distances between 1 nm and 4.5 nm 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 1.6–1.9 nm. 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 2 nm for DEER
measurements and this is calculated using the density matrix formalism.