Site‐Specific Isotopic Labeling (SSIL): Access to High‐Resolution Structural and Dynamic Information in Low‐Complexity Proteins

Remarkable technical progress in the area of structural biology has paved the way to study previously inaccessible targets. For example, large protein complexes can now be easily investigated by cryo‐electron microscopy, and modern high‐field NMR magnets have challenged the limits of high‐resolution characterization of proteins in solution. However, the structural and dynamic characteristics of certain proteins with important functions still cannot be probed by conventional methods. These proteins in question contain low‐complexity regions (LCRs), compositionally biased sequences where only a limited number of amino acids is repeated multiple times, which hamper their characterization. This Concept article describes a site‐specific isotopic labeling (SSIL) strategy, which combines nonsense suppression and cell‐free protein synthesis to overcome these limitations. An overview on how poly‐glutamine tracts were made amenable to high‐resolution structural studies is used to illustrate the usefulness of SSIL. Furthermore, we discuss the potential of this methodology to give further insights into the roles of LCRs in human pathologies and liquid–liquid phase separation, as well as the challenges that must be addressed in the future for the popularization of SSIL. Low‐complexity regions (LCRs) are strikingly simple sequences where only a limited number of amino acids are repeated. Although LCRs are quite common, their high‐resolution characterization is inherently difficult. Herein we present site‐specific isotopic labeling (SSIL) as a powerful tool to study these intriguing sequences and shed light on their structure–function relationships.