Soluble free radicals, such as the TEMPO nitroxide or its derivatives, have been used for spin-labelling of the solvent in NMR experiments. Thus, relevant structural information was obtained by observing the speeding up, due to the presence of the paramagnetic probe, of nuclear relaxation rates of proton nuclei of small and structured molecules from ID spectra. The complete absence of any change in the ESR spectrum of the nitroxide upon additions of gramicidin S first suggested a dynamic model where the solute conformation of a peptide in a TEMPO spin-labelled solvent governs the formation of weak collisional adducts between the nitroxide and the diamagnetic molecule. These dipolar electron-nucleus interactions determine the extent of the paramagnetic effects on nuclear relaxation rates. The same mechanism is also responsible of the peak intensity decrease in multidimensional spectra, as more recently has been proposed for peptides and proteins.

Here a guideline for a quantitative evaluation of the paramagnetic filters on the signal intensities of surface exposed nuclei is described. Data and analyses reported in literature on the subject (also using other conformational probes) is critically discussed.

The use of nitroxides probes seems to be a very promising technique, not only for solution structure determinations, but also for the characterisation of the folding / unfolding process due to the stability of the paramagnetic probe to pH, temperature, ionic strength and solvent changes, typical for this kind of studies.