Biarsenical- tetracysteine tagging system
The first tag developed for site-specific protein labeling was the tetracysteine motif, a short peptide usually composed of six amino acids CCPGCC that can bind with biarsenical dyes.36 The principle of the binding is the well-known interaction of arsenic with thiol groups. The first developed biarsenical dye, FlAsH, is a fluorescein derivative with two As(III) substituents, which is weakly fluorescent as an adduct with two 1,2-ethanedithiol (EDT) but becomes green fluorescent after binding with the tetracysteine tag (Figure 1.9).37,38 Analogs have been developed for expending the spectral properties.36,39 ReAsH (Figure 1.9) as the most useful one is a derivative of the red fluorophore resorufin and could be applied in pulsechase imaging with FlAsH.39 Although the small size of the peptide tag and the various biarsenical dyes makes it one of the best established site-specific labeling systems, it still needs to be improved in many aspects like the signal-to-background ratio, the binding specificity, the need of antidotes to reduce the toxicity and the requirement of a reducing environment.
Self-labeling tags: SNAP-tag, CLIP-tag, Halo-tag
SNAP-tag is a mutant of the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) developed by the group of Johnsson. 40 – 43 It can transfer the functionalized benzyl group of the O6-benzylguanine (BG) derivative to its active site cysteine and thus achieves covalent and irreversible labeling of fusion protein (Figure 1.10).
The versatility of the developed BG derivatives enables to easily adapt SNAP-tag functionalities to different conditions for visualizing proteins in vitro and in vivo.44-46 To meet the need for multiplexed imaging, the group of Johnsson developed CLIP-tag47, which shows substrate specificity orthogonal to SNAP-tag. CLIP-tag is an engineered mutant of AGT that works as SNAP-tag but reacts specifically with O2-benzylcytosine (BC) derivatives (Figure 1.10). These two tags have been used to label two different proteins in living cells for simultaneous pulse-chase experiments47 and also used to cross-link interacting proteins to detect and characterize protein-protein interactions48.
The Halo-tag is engineered from a bacterial halogen dehydrogenase. The carbon halogen bond of the functionalized chloroalkane derivative could be cleft by the Halo-tag and thus the fluorescent ligand covalently binds with the Halo-tag fusion protein (Figure 1.10).
Fluorogen-activating proteins (FAPs)
Fluorogen-activating proteins (FAPs) engineered from libraries of human single-chain antibodies (scFvs) bind non-covalently fluorogenic derivatives of malachite green (MG) and thiazole orange (TO) with nanomolar affinity and generate huge enhancement of their red and green fluorescence, respectively. 55 MG and TO are well-known molecular rotors that are poorly fluorescent in solution because of internal rotation; however in constrained environments, the internal rotations are slowed down, which strongly enhance their fluorescence intensities.54 FAPs were shown to be suitable to label proteins without the need of wash-out process. Cell impermeant MG-2p and TO1-2p enable labeling of only the FAPtagged proteins at the plasma membrane, while the cell permeant fluorogen MG-ester allows lighting up proteins in the secretory pathway within several minutes after addition (Figure 1.12).
Cellular retinoic acid binding protein II (CRABPII)
The cellular retinoic acid binding protein II (CRABPII) is a binding and transport protein that binds cellular retinoic acids and thus transport them inside the cell cytoplasm.67-69 Its small size (15.6 kDa), relatively large binding pocket that could binds with various synthetic retinoids (Figure 1.17) and tolerance of mutations makes it a promising protein scaffold for the engineering of fluorogenic fluorescent protein tag.70 The group of Borhan has developed a novel fluorogenic protein labeling system by reengineering CRABPII. A non-fluorescent precursor merocyanine aldehyde reacts with the active site Lys residue of CRABPII-tag to generate the fluorescent protonated Schiff base which also displays a huge red-shift of absorption and a significant increase of extinction coefficient compare to the precursor merocyanine aldehyde (Figure 1.18). The chromophore is planar and restricted in the protein cavity, which enhance its fluorescence.
Fluorescence-Activating and absorption-Shifting Tag (FAST)
Our group recently developed a novel fluorogenic protein labeling system called Fluorescence-Activating and absorption-Shifting Tag (FAST). 81 The fluorogenic dye 4- hydroxy-3-methylbenzylidene rhodanine HMBR binds with FAST non-covalently and generates a bright green-yellow fluorescent complex (Figure 1.21). In addition to fluorescence activation as generally observed with fluorogen-based labeling systems, HMBR also displays an absorption red shift after binding with FAST which enables high contrast imaging by distinguishing free and bound fluorogen via the choice of the excitation wavelength. This unique fluorogen activation mechanism implying two spectroscopic changes, fluorescence quantum yield increase and absorption red shift, ensures thus selective labeling and high imaging contrast..
Table of contents :
Chapter I General Introduction I-1 Fluorescence
1 I-1.1 The principle of fluorescence
1 I-1.2 Absorption, excitation and emission spectra
I-1.3 Fluorescence quantum yield, lifetime and brightness
I-1.4 Advantages of fluorescence-based investigative technologies
I-2 Fluorescent reporters
I-2.1 Organic fluorescent probes
I-2.2 Autofluorescent proteins (AFP)
I-2.2.1 Applications of AFPs
I-2.2.2 Limitations of AFPs
I-2.3 Site-specific labeling techniques
I-2.3.1 Biarsenical- tetracysteine tagging system
I-2.3.2 Self-labeling tags: SNAP-tag, CLIP-tag, Halo-tag
I-2.3.3 Fluorogenic site-specific labeling systems
I-2.4 Fluorogen-activating proteins (FAPs)
I-2.6 Cellular retinoic acid binding protein II (CRABPII)
I-2.8 Fluorescence-Activating and absorption-Shifting Tag (FAST)
Chapter II Expansion of the spectral properties of FAST
II-1 Presentation of article 1
II-2 Article 1: Dynamic multi-color protein labeling in living cells
Chapter III Development of far-red emitting FAST
III-1 Molecular engineering of far-red emitting fluorogens
III-1.1 Presentation of article 2
III-1.2 Article 2: Design and characterization of red fluorogenic push-pull chromophores holding great potential for bioimaging and biosensing.
III-2 Directed evolution of protein tags binding and activating far-red-emitting fluorogens
III-2.2 Results and discussions
III-2.3 Conclusion and perspective
III-2.4 Materials and Methods
Chapter IV Development of cell impermeant fluorogens for the selective imaging of cell surface proteins
IV-2 Results and discussion
IV-3 Conclusion and perspective
IV-4 Materials and Methods
Chapter V General discussion
V-1 Development of new fluorogens
V-2 Selection of new protein tags