Intermediate dipolar distances from spin labels.

Methods for determining inter-spin distances between nitroxide spin labels from dipolar couplings in the intermediate range (r12≈1.1-2 nm) by CW-EPR are addressed. For nitroxide powder patterns, the assumption of unlike spins is a better approximation than assuming strong coupling between like spins. Methods that determine the average splitting in dipolar deconvolutions yield the mean value 〈1/r12(3)〉, and those that determine the dipolar contribution to the spectral second moment correspondingly yield 〈1/r12(6)〉. To relate these ensemble averages to the mean inter-spin distance 〈r12〉 requires knowledge of the distribution in r12. Values of the inverse roots reff,n=〈1/r12(n)〉(-1/n) always lie below 〈r12〉. Consistent comparisons of literature data on double-labelled α-helical peptides support this view. Evaluations of reff,n for Gaussian distributions in r12 yield calibrations to determine 〈r12〉 for a given distribution width, σ(r12).