Determination of molecular alignment tensors without backbone resonance assignment: Aid to rapid analysis of...

Based on high-resolution structures of the free molecules accurate determination of structures of protein complexes by NMR spectroscopy is possible using residual dipolar couplings. In order, however, to be able to apply these methods, protein backbone resonances have to be assigned first. This NMR assignment process is particularly difficult and time consuming for protein sizes above 20 kDa. Here we show that, when NMR resonances belonging to a specific amino acid type are selected either by amino acid specific labeling, by their characteristic Calpha/Cbeta chemical shifts or by dedicated NMR experiments, molecular alignment tensors of proteins up to 80 kDa can be determined without prior backbone resonance assignment. This offers the opportunity to greatly accelerate determination of three-dimensional structures of protein-protein and protein-ligand complexes, and validation of multimeric states of proteins. Moreover, exhaustive back-calculation can be performed using only 1DNH couplings. Therefore, it avoids expensive 13C-labeling and it gives access to orientational information for large proteins that strongly aggregate at concentrations above 50 microM, i.e., experimental conditions where 3D triple resonance experiments are not sensitive enough to allow backbone resonance assignment.