A completely different approach to this topic was taken by Chen et al. [399,400]. Instead of using isotope labeling, Chen’s group decided to use a tert-butyl group contained within ligand-1 to obtain structural information about the protein-ligand complex [400]. The tert-butyl group formed an intense singlet in 1.0 to 1.5 ppm range thanks to rapid methyl rotation and methyl reorientation within that group. When compared with the protein’s 1H-NMR signal, the tert-butyl signal tended to be much narrower and resulted in easy detection without the need for isotopic enrichment even in protein complexes of high molecular mass such as Bacillus stearothermophilus DnaB hexamer (320 kDa) [399]. Additionally, the tert-butyl group produces intense NOESY cross peaks that can be observed even in the situations where normally cross-peaks of the proteins are barely detectable. This is partially because the signal corresponded to nine protons within tert-butyl group. Those aspects enable measurements of pseudo-contact shifts generated by paramagnetic tags attached to the protein. As a result, it allows positioning of the ligand on the protein. An example of this approach, is dengue virus NS2B-NS3 protease from serotype 2 (referred as DENpro) in complexed with ligand containing a tert-butyl group. The result of this experiment showed NOEs between the tert-butyl group of ligand-1 and residue Val155 from DENpro [400].