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

Nuclearmagnetic resonance (NMR) studies of large biomolecularmachines and highly repetitive proteins remain challenging due tothe difficulty of assigning frequencies to individual nuclei. Here,we present an efficient strategy to address this challenge by engineeringa Pyrococcus horikoshii tRNA/alanyl-tRNAsynthetase pair that enables the incorporation of up to three isotopicallylabeled alanine residues in a site-specific manner using in vitroprotein expression. The general applicability of this approach forNMR assignment has been demonstrated by introducing isotopically labeledalanines into four distinct proteins: huntingtin exon-1, HMA8 ATPase,the 300 kDa molecular chaperone ClpP, and the alanine-rich Phox2Btranscription factor. For large protein assemblies, our labeling approachenabled unambiguous assignments while avoiding potential artifactsinduced by site-specific mutations. When applied to Phox2B, whichcontains two poly-alanine tracts of nine and twenty alanines, we observedthat the helical stability is strongly dependent on the homorepeatlength. The capacity to selectively introduce alanines with distinctlabeling patterns is a powerful tool to probe structure and dynamicsof challenging biomolecular systems.

Automated summary

A strategy for efficiently assigning frequencies to individual nuclei in large biomolecular machines and repetitive proteins using in vitro protein expression is presented. The approach was demonstrated to be applicable for NMR assignment in four proteins by introducing isotopically labeled alanines. The labeling method enabled unambiguous assignments in large protein assemblies and revealed the correlation between helical stability and homorepeat length in Phox2B. Selectively introducing alanines with distinct labeling patterns proves to be a powerful tool for studying challenging biomolecular systems.