General discussion and future perspectives

The RE distance measurements still stay somewhat aside in this scheme. It is still an open question, what precision can be achieved in RE distance measurements in multiply-lahelled systems. RE measurements should also he done at X hand, while all the DEER measurements in Fig. 1(A) are best performed at higher detection frequencies. 

Nevertheless, Dy(iii)-nitroxide RE is a useful eomplementary technique, easily interchangeable with Gd(iii)-nitroxide DEER, and its importance should not be underestimated. 

The use of Cu(ii) or other transition metal centres as one of spectroscopically orthogonal labels for DEER measurements is complicated by the width of the corresponding ERR spectrum. A broad spectrum typically results in a strong selection of orientations and low modulation depth (if these species are pumped). While orientation selection can be useful, it clearly complicates the determination of inter-spin distance distributions. These problems might be resolved by employing pulses with very broad excitation bands, available for the pulse EPR setups based on arbitrary waveform generators. Recently such experiments were reported for Cu(ii)-nitroxide and Co(ii)-nitroxide spin pairs. °° It is worth mentioning recent work on DEER-based distance determination between nitroxide radicals and low-spin Fe(iii). °  

The Cu(ii) ions were also considered as possible RE agents. For the cases of Fe(iii) and Cu(ii) it is important to verify the applicabilify of the Redfield regime in the whole measured temperature range. The relaxation of these ions is typically slower than the one of Dy(iii) and deviations from the Redfield regime are possible at low temperatures. 

In a biomolecule, labelled with two Gd(m) labels and two nitroxide radicals, the measurement of Gd(iii)-nitroxide distances would in general produce an overlap of four distance distributions from all possible combinations of Gd(iii) and nitroxide labelling sites. Such measurements could provide important qualitative information, but their quantitative use might be limited. For instance the nitroxide-nitroxide and Gd(in)-Gd(iii) distances could be monitored in two different types of biomolecules and the distance changes upon complex formation could be further supported by the independent proof of the presence of the complex from Gd(iii)-nitroxide DEER. 

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