Small Molecule-Photoactive Yellow Protein Labeling Technology in Live Cell Imaging

Abstract

Characterization of the chemical environment, movement, trafficking and interactions of proteins in live cells is essential to understanding their functions. Labeling protein with functional molecules is a widely used approach in protein research to elucidate the protein location and functions both in vitro and in live cells or in vivo. A peptide or a protein tag fused to the protein of interest and provides the opportunities for an attachment of small molecule probes or other fluorophore to image the dynamics of protein localization. Here we reviewed the recent development of no-wash small molecular probes for photoactive yellow protein (PYP-tag), by the means of utilizing a quenching mechanism based on the intramolecular interactions, or an environmental-sensitive fluorophore. Several fluorogenic probes have been developed, with fast labeling kinetics and cell permeability. This technology allows quick live-cell imaging of cell-surface and intracellular proteins without a wash-out procedure.

Keywords: imaging; live cell; photo active yellow protein; protein labeling; small molecule.

Figure 1

General strategy for labeling protein with small molecules. Protein of interest (gray oval) fusion with peptide or protein tag (yellow oval), which is able to bind to a small molecule (blue oval). Upon binding, the detectable small molecule will give a certain fluorescent signal and protein function is analyzed by fluorescence microscopy or other detection methods.

Figure 2

Photoactive yellow protein structure (PDB: 2phy). The overall structure of PYP is divided into four segments: N-terminal cap (purple), contains residues form 1 to 28; PAS core (orange), where high sequence homology has been found among various PAS-containing molecules, contains residues from 29 to 69; Helical connector (green), spans from residue 70 to 87; β-scaffold (blue), spans from residues from 88 to 125, contains the last three strands of PYP.

Scheme 1

Crucial steps for biosynthesis of p-coumaryl: CoA. TAL: tyrosine ammonia lyase; pCl: p-coumaryl: coenzyme A ligase.

Figure 3

Principle of fluorogenic labeling system based on PYP.

Scheme 2

Structures of fluorescent probes CATP and FCTP.

Figure 4

Principle of fluorogenic probes with a turn on/off quencher.

Figure 5

Chemical structures of FCATP and FCANB.

Figure 6

Principle of the labeling system based on PYP-tag and environment-sensitive fluorogenic probe.

Figure 7

Chemical structures of TMBDMA and CMBDMA.

Figure 8

PYP-tag mutant PYP3R and its fluorogenic probeCMBDMA2 based on electrostatic interactions and the pK_a value of the leaving group.

Figure 9

Structures of AcFCANB and FCANB.