Site-Specific Introduction of Alanines for the NMR Investigation of Low-Complexity Regions and Large Biomolecular Assemblies

NMR studies of large biomolecular machines and highly repetitive proteins remain challenging due to the difficulty of assigning signals to individual nuclei. Here, we present an efficient strategy to address this challenge by engineering a Pyrococcus horikoshii tRNA/alanyl-tRNA synthetase pair that enables the incorporation of up to three isotopically labeled alanine residues in a site-specific manner using in vitro protein expression. We have demonstrated the general applicability of this approach for NMR assignment by introducing isotopically labeled alanines into four proteins, including the 300-kDa molecular chaperone ClpP and the alanine-rich Phox2B transcription factor. For large protein assemblies, our labeling approach enables unambiguous assignments, while avoiding potential artefacts induced by site-specific mutations. When applied to Phox2B, which contains two poly-alanine tracts of nine and twenty alanines, we observe that the helical stability is strongly dependent on the homorepeat length, demonstrating structural cooperativity. The capacity to selectively introduce alanines with distinct labeling patterns is a powerful tool to probe structure and dynamics of biomolecular systems that are out of the reach of traditional structural biology methods.