Simultaneous detection and deconvolution of congested NMR spectra containing three isotopically labeled species

We present a procedure for isolating subspectra corresponding to individual protein or peptide components in a ternary mixture or complex. Each of the three-component species is labeled differently: species A uniformly with ¹⁵N, species B uniformly with ¹⁵N and ¹³C, and species C uniformly with ¹⁵N but selectively with ¹³C′ or ¹³C^α. By using the dual carbon label selective HSQC (DCLS-HSQC) pulse sequence and exploiting differences in ¹J ¹⁵N-¹³C coupling patterns to filter selected ¹⁵N resonances from detection during a constant time period, a subspectrum from each species can be generated from three spectra acquired from a single sample. Many important biological pathways involve dynamic interactions among members of multicomponent protein assemblies, and this approach offers a powerful way to monitor such processes.