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

Abstract

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.

Evidence of the Reduced Abundance of Proline cis Conformation in Protein Poly Proline Tracts

“…To overcome the limitations encountered to probe the residuespecific cis/trans equilibrium within poly-P tracts in a native protein context, we applied the SSIL strategy. 40,41 This technique combines CF protein expression and nonsense suppression to site-specifically incorporate a single [ 15 N, 13 C]labeled amino acid into a protein, greatly simplifying NMR spectra. Here, we labeled proline residues within and outside of the poly-P tracts of H16.…”

Robust Cell-Free Expression of Sub-Pathological and Pathological Huntingtin Exon-1 for NMR Studies. General Approaches for the Isotopic Labeling of Low-Complexity Proteins

“…The investigation of proteins with low-complexity sequences represents a challenge for traditional structural biology approaches [ 27 , 44 ]. Due to their inherent flexibility, NMR is the most suited technique for these studies.…”

“…In addition to being aggregation prone, HttEx1 is highly flexible and crystallization and cryo-electron microscopy studies could not provide structural information on the poly-Q tract [ 21 , 24 ], leaving nuclear magnetic resonance (NMR) the only technique to obtain high-resolution data on HttEx1. However, with the repetitive nature of the Httex1 sequence, which produces highly congested spectra, traditional NMR experiments reach their limit, requiring novel strategies to support the sequential assignment and the acquisition of structural restraints [ 10 , 11 , 15 , 20 , 25 , 26 , 27 ].…”

Emerging Methods for Efficient and Extensive Incorporation of Non-canonical Amino Acids Using Cell-Free Systems

“…When aminoacylation and translation are conducted in the same system, in order to avoid competition between amino acids in the same aaRS/tRNA pair, the corresponding canonical amino acid should be removed to safeguard the orthogonality of the translation machine. Via pre-aminoacylation with ncAAs by aaRS, not only any codon, including stop codons, can be reassigned to a ncAA with a defined aminoacyl-tRNA, but also more efficient ncAA incorporation can be realized by optimizing additive concentrations ( Urbanek et al, 2020 ). For instance, the general method for the expression of isotopically labeled proteins relies on aaRS charging isotopically labeled amino acids.…”