Engineering of Tunable Allosteric-like Fluorogenic Protein Sensors.

Optical protein sensors that enable detection of relevant biomolecules of interest play central roles in biological research. Coupling fluorescent reporters with protein sensing units has enabled the development of a wide range of biosensors that recognize analytes with high selectivity. In these sensors, analyte recognition induces a conformational change in the protein sensing unit that can modulate the optical signal of the fluorescent reporter. Here, we explore various designs for the creation of tunable allosteric-like fluorogenic protein sensors through incorporation of sensing protein units within the chemogenetic fluorescence-activating and absorption-shifting tag (FAST) that selectively binds and stabilizes the fluorescent state of 4-hydroxybenzylidene rhodanine (HBR) analogs. Conformational coupling allowed us to design analyte-responsive optical protein sensors through allosteric-like modulation of fluorogen binding.

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

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.

An expanded palette of fluorogenic HaloTag probes with enhanced contrast for targeted cellular imaging.

We report the development of HaloTag fluorogens based on dipolar flexible molecular rotor structures. By modulating the electron donating and withdrawing groups, we have tuned the absorption and emission wavelengths to design a palette of fluorogens with emissions spanning the green to red range, opening new possibilities for multicolor imaging. The probes were studied in glycerol and in the presence of HaloTag and exhibited good fluorogenic properties thanks to a viscosity-sensitive emission. In live-cell confocal imaging, the fluorogens yielded only a very low non-specific signal that enabled wash-free targeted imaging of intracellular organelles and proteins with good contrast. Combining experimental studies and theoretical investigation of the protein/fluorogen complexes by molecular dynamics, these results offer new insight into the design of molecular rotor-based fluorogenic HaloTag probes in order to improve reaction rates and the imaging contrast.

Illuminating Cellular Biochemistry: Fluorogenic Chemogenetic Biosensors for Biological Imaging.

Cellular activity is defined by the precise spatiotemporal regulation of various components, such as ions, small molecules, or proteins. Studying cell physiology consequently requires the optical recording of these processes, notably by using fluorescent biosensors. The recent development of various fluorogenic systems greatly expanded the palette of reporters to be included in these sensors design. Fluorogenic reporters consist of a protein or RNA tag that can complex either an endogenous or a synthetic fluorogenic dye (so-called fluorogen). The intrinsic nature of these tags, along with the high tunability of their cognate chromophore provide interesting features such as far-red to near-infrared emission, oxygen independence, or unprecedented color versatility. These engineered photoreceptors, self-labelling proteins, or noncovalent aptamers and protein tags were rapidly identified as promising reporters to observe biological events. This Minireview focuses on the new perspectives they offer to design unique and innovative biosensors, thus pushing the boundaries of cellular imaging.