One-sample approach to determine the relative orientations of proteins in ternary and binary complexes from residual dipolar coupling measurements

(Figure Presented) We present a procedure that supports the acquisition of ¹H-¹⁵N residual dipolar coupling (RDC) values for individual subunits in binary or ternary protein assemblies from a single experimental sample. Our method relies on asymmetric labeling of each subunit with the following scheme: 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. Because only a single sample is required, the approach obviates the need for preparing multiple samples and eliminates potential errors introduced from differences in sample conditions. Because numerous biological processes rely on protein assemblies or transient interactions, this method should be well suited for a wide range of future applications.