Transient NOE-exchange-relay experiment: Application to ligand–protein binding under slow exchange conditions

Abstract

A new version of one-dimensional 1H experiment has been developed to probe ligand binding to macromolecular targets. The experiment, called transient NOE-exchange relay, is similar to the ‘reverse NOE pumping’ technique [A. Chen, M.J. Shapiro, J. Am. Chem. Soc. 122 (2000) 414–415]. The T 2 filter is used to erase protein magnetization, and the saturation then spreads from protein to bound ligand (via NOE) and further to a free ligand (via on–off exchange). The ligand signals, monitored as a function of mixing time, present a familiar ‘dip’ pattern characteristic of transient NOE or transient exchange experiments. In addition to the T 2 filter, we have also implemented a T 1 filter which makes use of the fact that the selective T 1 - 1 rates in macromolecules are much higher than those in small ligands. To model the experiment, complete relaxation and exchange matrix analysis has been invoked. This formalism was further used as a starting point to develop a simplified treatment where the relaxation and exchange components are represented by 2 × 2 matrix and, in addition, there is a special term responsible for coupling of ligand magnetization to the protein spin bath. The proposed experimental scheme has been tested on a system of peanut agglutinin complexed with Me-β- d-galactopyranoside, which is known to be in a slow exchange regime. The results suggest that the NOE-exchange-relay experiment can be used at the advanced stages of the drug development process to confirm high-affinity ligand binding.