Selective Two‐Step Labeling of Proteins with an Off/On Fluorescent Probe

Abstract

We present a novel design strategy for off/on fluorescent probes suitable for selective two-step labeling of proteins. To validate this strategy, we designed and synthesized an off/on fluorescent probe, 1-Ni(2+), which targets a cysteine-modified hexahistidine (His) tag. The probe consists of dichlorofluorescein conjugated with nitrilotriacetic acid (NTA)-Ni(2+) as the His-tag recognition site and a 2,4-dinitrophenyl ether moiety, which quenches the probe's fluorescence by photoinduced electron transfer (PeT) from the excited fluorophore to the 2,4-dinitrophenyl ether (donor-excited PeT; d-PeT) and also has reactivity with cysteine. His-tag recognition by the NTA-Ni(2+) moiety is followed by removal of the 2,4-dinitrophenyl ether quencher by proximity-enhanced reaction with the cysteine residue of the modified tag; this results in a marked fluorescence increase. Addition of His-tag peptide bearing a cysteine residue to aqueous probe solution resulted in about 20-fold fluorescence increment within 10 min, which is the largest fluorescence enhancement so far obtained with a visible light-excitable fluorescent probe for a His-based peptide tag. Further, we successfully visualized CysHis(6)-peptide tethered to microbeads without any washing step. The probe also showed a large fluorescence increment in the presence of His(6)Cys-tagged enhanced blue fluorescent protein (EBFP), but not His(6)-tagged EBFP. We consider this system is superior to large fluorescence tags (e.g., green fluorescent protein: 27 kDa), which can perturb protein folding, trafficking and function, and also to existing small tags, which generally show little fluorescence increase upon target recognition and therefore require a washout step. This strategy should also be applicable to other tags.

The Benefits of Unnatural Amino Acid Incorporation as Protein Labels for Single Molecule Localization Microscopy

“…In the context of proteins, a large component of successful SMLM imaging is the choice of fluorescent probe used as a label for the protein of interest (POI). The ideal fluorescent tag candidate for SMLM should aim for photostability during activation and deactivation cycles, be small in size, have high levels of photon emission for better single-molecule detection by the microscope and must not perturb the functionality of the intrinsic interactions of the POI and the other proteins in the environment (Hirabayashi et al, 2011;Toseland, 2013). Various approaches have been developed over the years and have seen great advancement in the creation of photoswitchable fluorophores (Zessin et al, 2012).…”

Labelling Bacterial Nanocages with Photo‐switchable Fluorophores

“…It has been proven that encapsulins can penetrate cells while carrying functional molecules,6 but further understanding of their in vivo behavior calls for the development of non‐invasive imaging and tracking strategies. Overcoming the diffraction barrier for microscopy can be achieved in live cells by using super‐resolution fluorescence imaging,10, 11, 12, 13, 14, 15, 16 by photo‐switching fluorescence on and off to statistically turn on a small fraction of fluorophores only 14, 15, 16, 17, 18, 19. The mapping of fluorescent points is achieved by activating only a subset of fluorophores stochastically at any given time while the rest are switched off 15.…”

“…However, despite their biocompatibility, they suffer drawbacks compared to synthetic fluorophores, which are often brighter than their biological counterparts,10 allow chemical modifications on surfaces22, 23 and are less likely to interfere with protein folding because they are smaller than fluorescent proteins 18. Consequently, synthetic and photo‐switchable fluorescent probes have emerged as powerful tools for in vivo imaging,21 including diarylethenes24 and spiropyran derivatives 25.…”

“…Overcoming the diffraction barrier for microscopy can be achieved in live cellsb yu sing super-resolution fluorescence imaging, [10][11][12][13][14][15][16] by photo-switching fluorescence on and off to statistically turn on as mall fraction of fluorophores only. [14][15][16][17][18][19] The mapping of fluorescent points is achieved by activating only as ubset of fluorophores stochastically at anygiven time while the rest are switched off. [15] Super-resolution images are then reconstructed from multiple time-resolved fluorescence images at different areas.…”

“…and are less likely to interfere with protein folding because they are smaller than fluorescent proteins. [18] Consequently, synthetic and photo-switchable fluorescent probes have emergeda sp owerful tools for in vivo imaging, [21] includingd iarylethenes [24] and spiropyran derivatives. [25] However,t heir moderate brightness remainsa ni ssue, whichi sw hy multiple fluorophores are often grafted at the surface of the same object.…”