Transverse relaxation optimized spectroscopy of NH2 groups in glutamine and asparagine side chains of proteins

A transverse relaxation optimized spectroscopy (TROSY) approach is described for the optimal detection of NH 2 groups in asparagine and glutamine side chains of proteins. Specifically, we have developed NMR experiments for isolating the slow-relaxing 15 N and 1 H components of NH 2 multiplets. Although even modest sensitivity gains in 2D NH 2 -TROSY correlation maps compared to their decoupled NH 2 –HSQC counterparts can be achieved only occasionally, substantial improvements in resolution of the NMR spectra are demonstrated for asparagine and glutamine NH 2 sites of a buried cavity mutant, L99A, of T4 lysozyme at 5 ºC. The NH 2 -TROSY approach is applied to CPMG relaxation dispersion measurements at the side chain NH 2 positions of the L99A T4 lysozyme mutant — a model system for studies of the role of protein dynamics in ligand binding.