Evaluation of the Cytosolic Uptake of HaloTag Using a pH-Sensitive Dye

Giancola, JoLynn B.; Grimm, Jonathan B.; Jun, Joomyung V.; Petri, Yana D.; Lavis, Luke D.; Raines, Ronald T.

doi:10.1021/acschembio.3c00713

Cited by 4 publications

(2 citation statements)

References 81 publications

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“…Live-cell fluorescence microscopy of HeLa cells treated with this conjugate showed cellular uptake (Figure B). In marked contrast, unconjugated HaloTag is not taken up by HeLa cells . To confirm that the protein construct was indeed delivered into the cytosol, we used an in-cell HiBiT–LgBiT complexation assay (Figure C).…”

Section: Resultsmentioning

confidence: 99%

Facile Access to Branched Multispecific Proteins

Okon,

Yang,

Giancola

et al. 2024

Bioconjugate Chem.

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Approaches that leverage orthogonal chemical reactions to generate protein−protein conjugates have expanded access to bespoke chimeras. Although the literature is replete with examples of the semisynthesis of bispecific proteins, few methods exist for the semisynthesis of protein conjugates of higher complexity (i.e., greater than two-protein fusions). The recent emergence of trispecific cell engagers for immune cell redirection therapies necessitates the development of chemical methods for the construction of trispecific proteins that would otherwise be inaccessible via natural protein synthesis. Here, we demonstrate that 3-bromo-5-methylene pyrrolone (3Br-5MP) can be used to effect the facile chemical synthesis of trispecific peptides and proteins with exquisite control over the addition of each monomer. The multimeric complexes maintain epitope functionality both in human cells and upon immobilization. We anticipate that facile access to trispecific proteins using this 3Br-5MP will have broad utility in basic science research and will quicken the pace of research to establish novel, multimeric immune cell redirection therapies.

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Section: Resultsmentioning

confidence: 99%

Facile Access to Branched Multispecific Proteins

Okon,

Yang,

Giancola

et al. 2024

Bioconjugate Chem.

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“…The ability to control the emission properties of fluorophores is crucial in the development of biosensors, imaging probes, fluorescence microscopy, and other optical tools for use in biological systems. − In the past 20 years, several innovative labeling strategies have emerged for cellular imaging, blending the genetic precision of proteins with the varied photophysical properties of small-molecule fluorophores. These include FlAsH, enzyme-based “self-labeling tags” (SNAP-, CLIP-, and Halo-Tag) − and electrophilic ligand–receptor pairs (coumarin–photoactive yellow protein, PYP). , Self-labeling systems like SNAP-, CLIP-, or Halo-tag have harnessed the flexibility of chemically synthesized fluorophores to create a range of fluorogenic, far- and infrared fluorophores. These advancements cater to the needs of super-resolution microscopy techniques and single molecule studies, offering enhanced versatility and precision in labeling strategies. ,,, …”

Section: Introductionmentioning

confidence: 99%

Regulation of Absorption and Emission in a Protein/Fluorophore Complex

Santos,

Chandra,

Assar

et al. 2024

ACS Chem. Biol.

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Human cellular retinol binding protein II (hCRBPII) was used as a protein engineering platform to rationally regulate absorptive and emissive properties of a covalently bound fluorogenic dye. We demonstrate the binding of a thio-dapoxyl analog via formation of a protonated imine between an active site lysine residue and the chromophore's aldehyde. Rational manipulation of the electrostatics of the binding pocket results in a 204 nm shift in absorption and a 131 nm shift in emission. The protein is readily expressed in mammalian systems and binds with exogenously delivered fluorophore as demonstrated by live-cell imaging experiments.

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A Fluorogenic Chemogenetic pH Sensor for Imaging Protein Exocytosis

Coïs,

Niepon,

Wittwer

et al. 2024

ACS Sens.

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Fluorescent protein-based pH biosensors enable the tracking of pH changes during protein trafficking and, in particular, exocytosis. The recent development of chemogenetic reporters combining synthetic fluorophores with self-labeling protein tags offers a versatile alternative to fluorescent proteins that combines the diversity of chemical probes and indicators with the selectivity of the genetic encoding. However, this hybrid protein labeling strategy does not avoid common drawbacks of organic fluorophores such as the risk of off-target signal due to unbound molecules. Here, we describe a novel fluorogenic and chemogenetic pH sensor based on a cell-permeable molecular pH indicator called pHluo-Halo-1, whose fluorescence can be locally activated in cells by reaction with HaloTag, ensuring excellent signal selectivity in wash-free imaging experiments. pHluo-Halo-1 was selected out of a series of four fluorogenic molecular rotor structures based on protein chromophore analogues. It displays good pH sensitivity with a pK a of 6.3 well-suited to monitor pH variations during exocytosis and an excellent labeling selectivity in cells. It was applied to follow the secretion of CD63-HaloTag fusion proteins using TIRF microscopy. We anticipate that this strategy based on the combination of a tunable and chemically accessible fluorogenic probe with a well-established protein tag will open new possibilities for the development of versatile alternatives to fluorescent proteins for elucidating the dynamics and regulatory mechanisms of proteins in living cells.

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Evaluation of the Cytosolic Uptake of HaloTag Using a pH-Sensitive Dye

Cited by 4 publications

References 81 publications

Facile Access to Branched Multispecific Proteins

Facile Access to Branched Multispecific Proteins

Regulation of Absorption and Emission in a Protein/Fluorophore Complex

A Fluorogenic Chemogenetic pH Sensor for Imaging Protein Exocytosis

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