Modulation of Fluorescent Protein Chromophores To Detect Protein Aggregation with Turn-On Fluorescence

• Published in: Journal of the American Chemical Society Vol. 140; no. 24; pp. 7381 - 7384
• Main Authors: Liu, Yu, Wolstenholme, Charles H, Carter, Gregory C, Liu, Hongbin, Hu, Hang, Grainger, Leeann S, Miao, Kun, Fares, Matthew, Hoelzel, Conner A, Yennawar, Hemant P, Ning, Gang, Du, Manyu, Bai, Lu, Li, Xiaosong, Zhang, Xin
• Format: Journal Article
• Language: English
• Published: WASHINGTON American Chemical Society 20.06.2018; Amer Chemical Soc
• Subjects: alpha-Synuclein - chemistry; alpha-Synuclein - metabolism; Chemistry; Chemistry, Multidisciplinary; Fluorescence; Fluorescent Dyes - chemical synthesis; Fluorescent Dyes - chemistry; HEK293 Cells; Humans; Huntingtin Protein - chemistry; Huntingtin Protein - metabolism; Imidazolines - chemical synthesis; Imidazolines - chemistry; Microscopy, Confocal; Microscopy, Fluorescence; Mutation; Physical Sciences; Protein Folding; Protein Multimerization; Science & Technology; Superoxide Dismutase-1 - chemistry; Superoxide Dismutase-1 - genetics; Superoxide Dismutase-1 - metabolism; BIOSENSORS; MODELS; LIVE-CELL; MIMICS; MISFOLDED PROTEIN; EMISSION; GREEN; GFP CHROMOPHORE; FLOW; LIVING CELLS
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/jacs.8b02176

We present a fluorogenic method to visualize misfolding and aggregation of a specific protein-of-interest in live cells using structurally modulated fluorescent protein chromophores. Combining photophysical analysis, X-ray crystallography, and theoretical calculation, we show that fluorescence is triggered by inhibition of twisted-intramolecular charge transfer of these fluorophores in the rigid microenvironment of viscous solvent or protein aggregates. Bioorthogonal conjugation of the fluorophore to Halo-tag fused protein-of-interests allows for fluorogenic detection of both misfolded and aggregated species in live cells. Unlike other methods, our method is capable of detecting previously invisible misfolded soluble proteins. This work provides the first application of fluorescent protein chromophores to detect protein conformational collapse in live cells.