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 N-15, species B uniformly with N-15 and C-13, and species C uniformly with N-15 but selectively with C-13' or C-13(alpha). By using the dual carbon label selective HSQC (DCLS-HSQC) pulse sequence and exploiting differences in (1)J(15)N-C-13 coupling patterns to filter selected N-15 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.