Observation of individual transitions in magnetically equivalent spin systems

Individual transitions of magnetically equivalent spin systems such as methyl groups residing on isotropically tumbling molecules in solution usually cannot be observed as multiplet-split NMR lines. We propose a pair of NMR experiments, 2D [13C,1Halphaalpha]Methyl and [13C,1Hbetabeta]Methyl HSQC, to overcome this limitation and enable direct and selective observation of individual 1H transitions in 13C-labeled methyl spin systems. Immediate applications include quantitative measurements of 1H-1H residual dipolar couplings (RDC) and cross-correlated relaxation between 1H chemical shift anisotropy and 1H-1H dipole-dipole interactions. The use of the experiments for the measurement of RDCs is demonstrated with two proteins, one weakly aligned by means of Pf1 phages and the other using a naturally present paramagnetic heme group.