A new method for the simultaneous measurement of magnitude and sign of D-1(CH) and D-1(HH) dipolar couplings in methylene groups

Heteronuclear dipolar couplings of the protein backbone have proven to have a big impact on the accuracy of protein NMR structures. H,H dipolar coupli ngs might have the same impact on side chains. Here we present a method tha t combines both heteronuclear and homonuclear dipolar couplings to investig ate the local conformation of methylene groups. A new pulse sequence (SPITZ E-HSQC) is presented, that allows to measure the two C,H and the H,H dipola r couplings at the same time, using spin state selective transfers. The new method has been applied to the methylene groups of glycines in the protein ubiquitin. The C,H and the H,H dipolar couplings might have a key role in fast stereospecific assignment of protons in CH2 groups.