Waterlogsy experiment

Principle :
WaterLOGSY is a widely applied 1D ligand-observation technique for the detection of protein–ligand interactions. As the STD approach, WaterLOGSY is based on the NOESY experiment, and implies transfer of magnetization via a intermolecular NOE and spin diffusion. The originality of WaterLOGSY comes from the intervention of water molecules in the transfer pathway. The bulk water magnetization is excited and transferred during the NOESY mixing time to the bound ligand via different mechanisms. The WaterLOGSY spectrum, which is recorded for the free ligand, contains the bound-state perturbed magnetization as long as the relaxation time T1 of the ligand is greater than the dissociation rate constant koff. The inverted water magnetization can be transferred via different pathways to the bound ligand:

(1) direct transfer from water molecules immobilized in the protein
binding site (water residence times greater than nanoseconds)

(2) chemical exchange between excited water and protein labile protons (amide, hydroxyl, amino, etc.) and propagation of the inverted magnetization to the ligand by intermolecular dipole–dipole crossrelaxation as well as spin diffusion via the protein– ligand complex

(3) transfer from the water molecules found in the protein surface via the protein–ligand complex. In the three mechanisms, the ligands interact with water via water–ligand–protein or protein–ligand complexes, whose rotational correlation times yield negative cross-relaxation rates and exhibit a negative NOE with water. By contrast, small molecules that only interact with bulk water (non-binders) will experience much faster tumbling, which translates into a positive NOE. Therefore, opposite signs for signals from free versus protein-bound ligands are observed in a WaterLOGSY spectrum, which enables one to easily discriminate binders and non-binders.

 waterlogsy-principle Waterlogsy-spectre

Experimental conditions:

For small proteins (molecular mass below 30 kDa), a concentration of 100 nM is required, whereas 1 µM is sufficient for larger proteins (molecular mass greater than 60 kDa). The ratio should usually not exceed 100:1, with the ligand concentration classically ranging between 40 µM and several hundred micromoles per litre. In general, mixing times reported in the literature range from 1 to 3 s. The temperature of the experiment can be decreased to 15 °C for low molecular weight receptors to artificially favour the spin diffusion through the protein.