Fluorogenic Photoaffinity Labeling of Proteins in Living Cells

Abstract

Genetically encoded fluorescent proteins or small-molecule probes that recognize specific protein binding partners can be used to label proteins to study their localization and function with fluorescence microscopy. However, these approaches are limited in signal-to-background resolution and the ability to temporally control labeling. Herein, we describe a covalent protein labeling technique using a fluorogenic malachite green probe functionalized with a photoreactive crosslinker. This enables a controlled covalent attachment to a genetically encodable fluorogen activating protein (FAP) with low background signal. We demonstrate covalent labeling of a protein in vitro as well as in live mammalian cells. This method is straightforward, displays high labeling specificity, and results in improved signal-to-background ratios in photoaffinity labeling of target proteins. Additionally, this probe provides temporal control over reactivity, enabling future applications in real-time monitoring of cellular events.

Rational Design of Bioavailable Photosensitizers for Manipulation and Imaging of Biological Systems

“…We set out to develop a hybrid small molecule:protein photosensitizer system that combines the flexibility and performance of small-molecule dyes with the subcellular specificity of genetically encoded reagents. To complement the recent development of impressive noncovalent hybrid photosensitizer systems (He et al, 2016;Marek and Davis, 2002;Tour et al, 2003) based on labeling strategies that require a constant supply of smallmolecule ligand (Ayele et al, 2019), we aimed to use the covalent HaloTag-labeling system developed by Promega (Los et al, 2008). This ''self-labeling'' enzyme-derived tag system has been used in many contexts ranging from single cells (Chong et al, 2018) to animals (Abdelfattah et al, 2019) and provides rapid, specific labeling of protein fusions with synthetic fluorophores.…”

Covalent Probes for Aggregated Protein Imaging via Michael Addition

“…To demonstrate this chemistry reported here can be utilized to develop other fluorescence color‐switch probes, we designed MG probe (P4) based on the scaffold of malachite green (MG) dye (Figure 6 a). MG derivatives have been reported to be fluorogenic upon binding to evolved peptide sequences [50, 51] . Thus MG scaffold may inherently bind to aggregated proteins.…”

“…MG derivatives have been reported to be fluorogenic upon binding to evolved peptide sequences. [50,51] Thus MG scaffold may inherently bind to aggregated proteins.MGofpositive charge possesses electropositivity in the center of the molecule and has been reported as ar eversible Michael acceptor with thiol group (Figure S16b). [52] Encouraged by these pieces of evidence,w e tested the fluorescence color-switch property of P4 upon the aggregation of mut-DHFR.…”

Identification of photocrosslinking peptide ligands by mRNA display

“…They can be used to identify new drug targets/off-target interactions, [1][2][3][4] to uncover the structure and location of drug binding sites, 5,6 for covalent protein labelling or for selective protein immobilisation. [7][8][9] Photoaffinity probes typically consist of a photoactivatable functionality appended to a targetselective ligand. Upon exposure to light, the photoactivatable moiety irreversibly crosslinks to the target of interest.…”