Multiexcitation Fluorogenic Labeling of Surface, Intracellular, and Total Protein Pools in Living Cells

Abstract

Malachite green (MG) is a fluorogenic dye that shows fluorescence enhancement upon binding to its engineered cognate protein, a fluorogen activating protein (FAP). Energy transfer donors such as cyanine and rhodamine dyes have been conjugated with MG to modify the spectral properties of the fluorescent complexes, where the donor dyes transfer energy through Förster resonance energy transfer to the MG complex resulting in binding-conditional fluorescence emission in the far-red region. In this article, we use a violet-excitable dye as a donor to sensitize the far-red emission of the MG-FAP complex. Two blue emitting fluorescent coumarin dyes were coupled to MG and evaluated for energy transfer to the MG-FAP complex via its secondary excitation band. 6,8-Difluoro-7-hydroxycoumarin-3-carboxylic acid (Pacific blue, PB) showed the most efficient energy transfer and maximum brightness in the far-red region upon violet (405 nm) excitation. These blue-red (BluR) tandem dyes are spectrally varied from other tandem dyes and are able to produce fluorescence images of the MG-FAP complex with a large Stokes shift (>250 nm). These dyes are cell-permeable and are used to label intracellular proteins. Used together with a cell-impermeable hexa-Cy3-MG (HCM) dye that labels extracellular proteins, we are able to visualize extracellular, intracellular, and total pools of cellular protein using one fluorogenic tag that combines with distinct dyes to effect different spectral characteristics.

Light-Up “Channel Dyes” for Haloalkane-Based Protein Labeling in Vitro and in Bacterial Cells

“…17 Naganbabu et al were able to develop probes using a FRET strategy based on the malachite green/fluorogen activating protein pair, with 66- and 417-fold light-up responses and Stokes shifts larger than 250 nm. 18 For the HaloTag enzyme, there exist several mentions in the literature of light-up substrates. In the first, Lukinavičius et al report a silicon-rhodamine probe used in super-resolution microscopy that exhibits a 6-fold increase in fluorescence upon reaction with the HaloTag domain.…”

Fluorogen activating protein toolset for protein trafficking measurements

“…Labeling all tagged protein (inside and out) can be a useful tactic for relating the amount of protein at the cell surface with total amount of protein, distinguishing proteostatic or biosynthetic effects from trafficking effects (Figure B). Either a matched or a different color cell‐permeable dye can be used sequentially after surface labeling to give a surface/total POI measurement that increases overall mechanistic insight in a variety of multi‐cell or single‐cell assays (Figure E) …”

“…Either a matched or a different color cell-permeable dye can be used sequentially after surface labeling to give a surface/total POI measurement that increases overall mechanistic insight in a variety of multi-cell or single-cell assays ( Figure 2E). [25][26][27] The FAP platform was developed by screening a yeast display library of single chain variable fragment antibodies (scFv) to find proteins that bind and activate two distinct cell-excluded fluorogenic dye derivatives: a malachite green with an amido-diethylene glycol amine linker (MG-2p) and a sulfonated thiazole orange with a similar linker (TO1-2p). The dL5** and AM2-2 FAP tags noncovalently bind MG and TO1 dye derivatives respectively with high affinities (Figures 3 and 4).…”

Fluorogenic Protein‐Based Strategies for Detection, Actuation, and Sensing

“…By using a cell‐permeant and an cell‐impermeant fluorogen the dynamics of BK channel plasma membrane residency was examined . This system was further extended by conjugation of a coumarin to a malachite green‐based fluorogen and co‐labeling with a cell‐impermeant malachite green fluorogen yielded a three‐color, two‐FRET read‐out of the extracellular, intracellular, and total protein pools . This concept has also been demonstrated with FAST, where labeling with a cell‐permeant and impermeant fluorogen allowed for the assessment of small molecule effects on GPCR trafficking using flow cytometry .…”

“…[64,65] This system was further extended by conjugation of a coumarin to a malachite green-based fluorogen and co-labeling with a cell-impermeant malachite green fluorogen yielded a three-color, two-FRET readout of the extracellular, intracellular, and total protein pools. [66] This concept has also been demonstrated with FAST, where labeling with a cell-permeant and impermeant fluorogen allowed for the assessment of small molecule effects on GPCR trafficking using flow cytometry. [67] While non-covalent systems such as the FAPs tightly bind their fluorogens to generate stable complexes with slow off rates and function similarly to classic fluorescent proteins and self-labeling systems, systems that exhibit dynamic exchange with their cognate fluorogens can be [92] EcFbFP FMN Non-covalent Dimer 448 496 14 500 44 [92] PpFbFP FMN Non-covalent Dimer 450 496 13 900 27 [92] iLOV FMN Non-covalent Monomer 447 497 44 [19] phiLOV2.1 FMN Non-covalent Monomer 450 497 20 [92] miniSOG FMN Non-covalent Monomer 447 497 14 200 41 170 pM [92] IFP1.4 Biliverdin Covalent Dimer 684 708 92 000 7.0 [21] iRFP Biliverdin Covalent Dimer 692 713 105 000 5.9 [22] iRFP670 Biliverdin Covalent Dimer 643 670 114 000 11.1 [25] iRFP682 Biliverdin Covalent Dimer 663 682 90 000 11.3 [25] iRFP702 Biliverdin Covalent Dimer 673 702 93 000 8.2 [25] iRFP720 Biliverdin Covalent Dimer 702 720 96 000 6.0 [25] IFP2.0 Biliverdin Covalent Dimer 690 711 98 000 8.1 [23] mIFP Biliverdin Covalent Monomer 683 705 82 000 8.4 [24] miRFP670 Biliverdin Covalent Monomer 642 670 87 400 14 [26] miRFP703 Biliverdin Covalent Monomer 674 703 90 900 8.6 [26] miRFP709 Biliverdin Covalent Monomer 683 709 78 400 5.4 [26] smURFP Biliverdin Covalent Dimer 642 670 180 000 18 [27] BDFP1.1 Biliverdin Covalent Dimer 682 707 68 700 5.9 [28] BDFP1.5 Biliverdin Covalent Monomer 688 711 74 000 5.0 [28] UnaG Bilirubin Non-covalent Monomer 498 527 77 300 51 98 pM [20] FAP HL1.01 Thiazole Orange Non-covalent Monomer 509 530 60 000 47 1.7 nM [41] FAP...…”