Kinetic and Structural Characterization of the Self-Labeling Protein Tags HaloTag7, SNAP-tag, and CLIP-tag

Wilhelm, Jonas; Kühn, Stefanie F.; Tarnawski, Mirosław; Gotthard, Guillaume; Tünnermann, Jana; Tänzer, Timo; Karpenko, Iuliia A.; Mertes, Nicole; Xue, Lin; Uhrig, Ulrike; Reinstein, Jochen; Hiblot, Julien; Johnsson, Kai

doi:10.1021/acs.biochem.1c00258

Cited by 86 publications

(122 citation statements)

References 69 publications

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“…The rhodamine binding site of HaloTag7 is formed by three helices (6–8), as revealed by the crystal structure of tetramethyl-rhodamine (TMR) bound to HaloTag7 (Protein Data Bank (PDB) identification number (ID): 6Y7A , 1.4 Å) 17 . To engineer the binding site, ten amino acids on helices 6–8 in close proximity to TMR were chosen for randomization by site-saturation mutagenesis (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Engineered HaloTag variants for fluorescence lifetime multiplexing

et al. 2021

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Self-labeling protein tags such as HaloTag are powerful tools that can label fusion proteins with synthetic fluorophores for use in fluorescence microscopy. Here we introduce HaloTag variants with either increased or decreased brightness and fluorescence lifetime compared with HaloTag7 when labeled with rhodamines. Combining these HaloTag variants enabled live-cell fluorescence lifetime multiplexing of three cellular targets in one spectral channel using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. Additionally, the brightest HaloTag variant showed up to 40% higher brightness in live-cell imaging applications.

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Section: Resultsmentioning

confidence: 99%

“…113916) 26 , Ig-κ-/PDGFR 27 and SNAPf (Addgene plasmid no. 167271) 17 were available inhouse and used as template plasmids. mCherry-LaminB1-10 was a gift from M. Davidson (Addgene plasmid no.…”

Section: Methodsmentioning

confidence: 99%

Engineered HaloTag variants for fluorescence lifetime multiplexing

et al. 2021

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“…For SiR, conjugation to HaloTag resulted in ∼3× larger K and t on in ROXS PCD compared with SNAP-tag in the same buffer. It was recently reported that fluorophores conjugated to HaloTag are tightly associated to the protein surface, while fluorophores conjugated to SNAP-tag protrude away from the protein surface ( Wilhelm et al. , 2021 ).…”

Section: Resultsmentioning

confidence: 99%

Photobleaching step analysis for robust determination of protein complex stoichiometries

Hummert

Yserentant

Fink

et al. 2021

MBoC

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The counting of discrete photobleaching steps in fluorescence microscopy is ideally suited to study protein complex stoichiometry in situ. The counting range of photobleaching step analysis has significantly improved with more sophisticated algorithms for step detection, albeit at an increasing computational cost and with the necessity for high data quality. Here, we address concerns regarding robustness, automation, and experimental validation, optimizing both data acquisition and analysis. To make full use of the potential of photobleaching step analysis, we evaluate various labelling strategies with respect to their molecular brightness, photostability, and photoblinking. The developed analysis algorithm focuses on automation and computational efficiency. Moreover, we validate the developed methods with experimental data acquired on DNA origami labeled with defined fluorophore numbers, demonstrating counting of up to 35 fluorophores. Finally, we show the power of the combination of optimized trace acquisition and automated data analysis by counting labeled nucleoporin 107 in nuclear pore complexes of intact U2OS cells. The successful in situ application promotes this framework as a new resource enabling cell biologists to robustly determine the stoichiometries of molecular assemblies at the single-molecule level in an automated fashion.

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“…These chemistry-backed advances are often developed hand-in-hand with molecular biology tools, a classic example being the 'magic bullet for kinases' -the engineering of the 'gatekeeper' residue in the catalytic pocket as a generic approach to inhibit the enzymatic activity of virtually any protein kinase of interest. [1] Subsequent examples include the generation of genetically-encoded, self-labeling protein tags such as the Halo, SNAP and CLIP tags, [2] and rapamycin-based protein dimerization systems such as the anchor-away, [3] or knock-sideways systems. [4] Optogenetics [5] represents an important variation on this chemical biology theme wherein cellular activities are manipulated by light rather than small molecules.…”

Section: Sensing Of Membrane Tensionmentioning

confidence: 99%

Chemical-Biology-derived in vivo Sensors: Past, Present, and Future

Loewith

Roux

Pertz

2021

Chimia

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To understand the complex biochemistry and biophysics of biological systems, one needs to be able to monitor local concentrations of molecules, physical properties of macromolecular assemblies and activation status of signaling pathways, in real time, within single cells, and at high spatio-temporal resolution. Here we look at the tools that have been / are being / need to be provided by chemical biology to address these challenges. In particular, we highlight the utility of molecular probes that help to better measure mechanical forces and flux through key signalling pathways. Chemical biology can be used to both build biosensors to visualize, but also actuators to perturb biological processes. An emergent theme is the possibility to multiplex measurements of multiple cellular processes. Advances in microscopy automation now allow us to acquire datasets for 1000's of cells. This produces high dimensional datasets that require computer vision approaches that automate image analysis. The high dimensionality of these datasets are often not immediately accessible to human intuition, and, similarly to 'omics technologies, require statistical approaches for their exploitation. The field of biosensor imaging is therefore experiencing a multidisciplinary transition that will enable it to realize its full potential as a tool to provide a deeper appreciation of cell physiology.

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Kinetic and Structural Characterization of the Self-Labeling Protein Tags HaloTag7, SNAP-tag, and CLIP-tag

Cited by 86 publications

References 69 publications

Engineered HaloTag variants for fluorescence lifetime multiplexing

Engineered HaloTag variants for fluorescence lifetime multiplexing

Photobleaching step analysis for robust determination of protein complex stoichiometries

Chemical-Biology-derived in vivo Sensors: Past, Present, and Future

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