Transverse relaxation optimized spectroscopy of NH groups in glutamine and asparagine side chains of proteins.

A transverse relaxation optimized spectroscopy (TROSY) approach is described for the optimal detection of NH groups in asparagine and glutamine side chains of proteins. Specifically, we have developed NMR experiments for isolating the slow-relaxing N and H components of NH multiplets. Although even modest sensitivity gains in 2D NH-TROSY correlation maps compared to their decoupled NH-HSQC counterparts can be achieved only occasionally, substantial improvements in resolution of the NMR spectra are demonstrated for asparagine and glutamine NH sites of a buried cavity mutant, L99A, of T4 lysozyme at 5 ºC. The NH-TROSY approach is applied to CPMG relaxation dispersion measurements at the side chain NH positions of the L99A T4 lysozyme mutant - a model system for studies of the role of protein dynamics in ligand binding.