Noncanonical amino acid incorporation enables minimally disruptive labeling of stress granule and TDP-43 proteinopathy

Abstract We report a minimally disruptive labeling strategy for stress granule protein, G3BP Stress Granule Assembly Factor 1 (G3BP1), and ALS-linked protein, TAR DNA-binding protein 43 (TDP-43), using the fluorescent noncanonical amino acid Anap. By integrating genetic code expansion (GCE) with rational site selection, we achieved precise incorporation of Anap that preserves protein structure and function. In live cells and neurons, Anap labeling faithfully recapitulated localization, stress-induced dynamics, and recovery behavior, outperforming conventional fluorescent tags and enabling physiologically relevant visualization of protein pathobiology. Competing Interest Statement The authors have declared no competing interest. Footnotes This revision addresses all reviewer comments and suggestions, with the inclusion of new data, expanded analyses, and refined descriptions that enhance clarity and strengthen the study.