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Article Dans Une Revue Analytical Chemistry Année : 2023

Genetic Targeting of Solvatochromic Dyes for Probing Nanoscale Environments of Proteins in Organelles

Résumé

A variety of protein tags are available for genetically encoded protein labelling, which allow their precise localization and tracking inside the cells. A new dimension in protein imaging can be offered by combining protein tags with polarity-sensitive fluorescent probes, which provide information about local nanoscale environment of target protein within the subcellular compartments (organelles). Here, we designed three fluorescent probes based on a solvatochromic Nile Red dye, conjugated to a HaloTag reactive targeting group through PEG linkers of varying length. The probe with medium linker length, NR12-Halo, was found to label specifically a large variety of proteins localized in defined cell compartments, such as plasma membranes (outer and inner leaflets), endoplasmic reticulum (ER), Golgi Apparatus, cytosol, microtubules, actin and chromatin. Owing to polarity-sensitive fluorophore, the probe clearly distinguished the proteins localized within apolar lipid membranes from other proteins. Moreover, it revealed dramatic changes in the environment during the life cycle of proteins from biosynthesis to their expected localization and finally, to recycling inside lysosomes. Heterogeneity in the local polarity of some membrane proteins also suggested a formation of low-polar protein aggregates, for example within cell-cell contacts. The approach also showed that mechanical stress (cell shrinking by osmotic shock) induced general polarity decrease in membrane proteins, probably due to condensation of biomolecules. Finally, the nano-environment of some membrane proteins was affected by polyunsaturated fatty acid (PUFA) diet, which provided the bridge between organization of lipids and proteins. The developed solvatochromic HaloTag probe constitutes a promising tool for probing nanoscale environment of proteins and their interactions within subcellular structures.
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Dates et versions

hal-04305283 , version 1 (24-11-2023)

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Rémi Pelletier, Dmytro Danylchuk, Hela Benaissa, Fanny Broch, Romain Vauchelles, et al.. Genetic Targeting of Solvatochromic Dyes for Probing Nanoscale Environments of Proteins in Organelles. Analytical Chemistry, 2023, 95 (22), pp.8512-8521. ⟨10.1021/acs.analchem.3c00515⟩. ⟨hal-04305283⟩
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