New NMR Methods for the Characterization of Bound Waters in Macromolecules

The study of water molecules which are associated with macromolecules, either at the surface or buried within the interior, is important because these waters play significant structural, catalytic, and/or recognition roles. We demonstrate new NMR techniques that will allow the determination of the lifetimes of certain bound water molecules. These techniques employ (i) pulsed field gradients (PFG) for diffusion editing, (ii) isotope editing for selective detection and solvent suppression, and (iii) selective excitation for efficiency of acquisition. Using a uniformly ¹⁵N-labeled fragment of the Escherichia coli chaperone protein, DnaJ, we show that a range of exchange lifetimes is observed for protons that physically exchange between water and amide sites. These methods will similarly allow the differentiation of bound water molecules on the basis of their lifetimes in the bound state and will be of general utility in the future for detailed studies of the dynamics of bound waters that have lifetimes in the 100 µs to 10 ms range.