Separation of intramolecular NOE and exchange peaks in water exchange spectroscopy using spin-echo filters

A technique for separating intramolecular NOE and solvent-proton exchange peaks in exchange spectroscopy is demonstrated. This method utilizes the large differences in relaxation and coupling properties of water and macromolecules to separate the two effects. The spin-echo filter consists of a water-frequency selective 90 degrees pulse followed by a spin-echo sequence. If the echo time is sufficiently long, protein resonances (e.g. C alpha H protons) excited by the selective pulse are removed due to their much shorter T2 values and J-coupling evolution. By combining the filter with exchange spectroscopy (EXSY) or water exchange (WEX) filter experiments, exchange peaks can be selectively observed. In this paper the filter is combined with a modified version of the WEX filter (WEX II filter) with 1D and 2D detection and applied to a zinc finger peptide and to staphylococcal nuclease, allowing estimation of the contribution of intramolecular NOEs to the exchange spectra.