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

化学 细胞器 内质网 荧光团 生物物理学 高尔基体 膜蛋白 细胞生物学 连接器 生物化学 荧光 生物 操作系统 物理 量子力学 计算机科学
作者
Rémi Pelletier,Dmytro I. Danylchuk,Hela Benaissa,Fanny Broch,Romain Vauchelles,Arnaud Gautier,Andrey S. Klymchenko
出处
期刊:Analytical Chemistry [American Chemical Society]
卷期号:95 (22): 8512-8521 被引量:9
标识
DOI:10.1021/acs.analchem.3c00515
摘要

A variety of protein tags are available for genetically encoded protein labeling, 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 environments of target proteins within the subcellular compartments (organelles). Here, we designed three fluorescent probes based on solvatochromic nile red dye, conjugated to a HaloTag reactive targeting group through polyethylene glycol linkers of varying lengths. 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, Golgi apparatus, cytosol, microtubules, actin, and chromatin. Owing to its 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 a general polarity decrease in membrane proteins, probably due to the condensation of biomolecules. Finally, the nanoenvironment of some membrane proteins was affected by a polyunsaturated fatty acid diet, which provided the bridge between organization of lipids and proteins. The developed solvatochromic HaloTag probe constitutes a promising tool for probing nanoscale environments of proteins and their interactions within subcellular structures.
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