Accessibility-dependent topology studies of membrane proteins using a SpyTag/SpyCatcher protein-ligation system

Bae, Yoonji; Lee, Sang Kwon; Chae, Young Chan; Park, Chan Young; Kang, Sebyung

doi:10.1016/j.ijbiomac.2021.02.015

Cited by 5 publications

(2 citation statements)

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“…More than 800 constructs have been published with SpyTag/SpyCatcher or its relatives at terminal sites on proteins, but there is little study of performance in protein loops ( Keeble and Howarth, 2019 ). Fusion of SpyTag in a loop of the multipass membrane protein Orai1 indicated that SpyCatcher reaction was inefficient in this context ( Bae et al., 2021 ). Probing the utility of SpyTag003 for reaction in protein loops, we show that SpyTag003 reaction with SpyCatcher003 was dramatically slowed within loops.…”

Section: Introductionmentioning

confidence: 99%

DogCatcher allows loop-friendly protein-protein ligation

Keeble

Yadav

Ferla

et al. 2022

Cell Chemical Biology

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There are many efficient ways to connect proteins at termini. However, connecting at a loop is difficult because of lower flexibility and variable environment. Here, we have developed DogCatcher, a protein that forms a spontaneous isopeptide bond with DogTag peptide. DogTag/DogCatcher was generated initially by splitting a Streptococcus pneumoniae adhesin. We optimized DogTag/DogCatcher through rational design and evolution, increasing reaction rate by 250-fold and establishing millimolar solubility of DogCatcher. When fused to a protein terminus, DogTag/DogCatcher reacts slower than SpyTag003/Spy-Catcher003. However, inserted in loops of a fluorescent protein or enzyme, DogTag reacts much faster than SpyTag003. Like many membrane proteins, the ion channel TRPC5 has no surface-exposed termini. DogTag in a TRPC5 extracellular loop allowed normal calcium flux and specific covalent labeling on cells in 1 min. DogTag/DogCatcher reacts under diverse conditions, at nanomolar concentrations, and to 98% conversion. Loop-friendly ligation should expand the toolbox for creating protein architectures.

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Section: Introductionmentioning

confidence: 99%

DogCatcher allows loop-friendly protein-protein ligation

Keeble

Yadav

Ferla

et al. 2022

Cell Chemical Biology

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“…Further, the reaction occurs across a broad range of conditions and circumvents issues with non-specific interactions by virtue of the covalent bond formed with the cognate protein binding. More recently, SpyCatcher has been used for fluorescence and single molecule localization imaging in bacteria and mammalian cells [16][17][18][19][20].…”

Section: Introductionmentioning

confidence: 99%

Sneaking in SpyCatcher using cell penetrating peptides for in vivo imaging

2023

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In vivo imaging of protein complexes is a powerful method for understanding the underlying biological function of these key biomolecules. Though the engineering of small, high affinity nanobodies have become more prevalent, the off-rates of these tags may result in incomplete or partial labeling of proteins in live cells. The SpyCatcher003 and SpyTag split protein system allow for irreversible, covalent binding to a short target peptide unlike nanobody-affinity based probes. However, delivering these tags into a cell without disrupting its normal function is a key challenge. Cell Penetrating Peptides (CPPs) are short peptide sequences that facilitate the transduction of otherwise membrane-impermeable “cargo” , such as proteins, into cells. Here we report on our efforts to engineer and characterize CPP-SpyCatcher003 fusions as modular imaging probes. We selected three CPPs, CUPID, Pentratin, and PVEC, to engineer fusion protein probes for superresolution microscopy, with the aim to eliminate prior permeabilization treatments that could introduce imaging artifacts. We find that fusing the CPP sequences to SpyCatcher003 resulted in dimer and multimer formation as determined by size exclusion chromatography, dynamic light scattering, and SDS resistant dimers on SDS-PAGE gels. By isolating and labeling the monomeric forms of the engineered protein, we show these constructs retained their ability to bind SpyTag and all three CPP sequences remain membrane active, as assessed by CD spectroscopy in the presence of SDS detergent. Using fluorescence and super resolution Lattice Structured Illumination Microscopy (Lattice SIM) imaging we show that the CPPs did not enhance uptake of SpyCatcher by E. coli, however with Caulobacter crescentus cells, we show that Penetratin, and to a lesser degree CUPID, does enhance uptake. Our results demonstrate the ability of the CPP-SpyCatcher003 to label targets within living cells, providing the groundwork for using split protein systems for targeted in vivo imaging.

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