Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%

Goedhart, Joachim; Stetten, David von; Noirclerc‐Savoye, Marjolaine; Lelimousin, Mickaël; Joosen, Linda; Hink, Mark A.; Weeren, Laura van; Gadella, Theodorus W. J.; Royant, Antoine

doi:10.1038/ncomms1738

Cited by 688 publications

(701 citation statements)

References 36 publications

(70 reference statements)

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“…These split constructs have allowed us to obtain two-color or super-resolution images of endogenous proteins and have revealed ER tubules with greatly reduced abundance of the translocon component Sec61B. Our platform can be easily extended to the engineering of other self-complmenting split FPs with distinct colors (e.g., mTurquoise2, mTagBFP2) 28,29 , good photoactivation performance (e.g., mMaple3, PATagRFP) 30,31 , or new functionalities (e.g., pH sensitivity) 32 . We note that for non-selfcomplementing split FPs, which are used to detect protein-protein interactions in bimolecular fluorescence complementation (BiFC) assays 33 , a different engineering platform is needed to ensure minimum affinity between the two FP fragments by themselves.…”

Section: Discussionmentioning

confidence: 99%

Improved Split Fluorescent Proteins for Endogenous Protein Labeling

Feng

Sekine

Pessino

et al. 2017

Preprint

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Self-complementing split fluorescent proteins (FPs) have been widely used for protein labeling, visualization of subcellular protein localization, and detection of cell-cell contact.To expand this toolset, we have developed a screening strategy for the direct engineering of self-complementing split FPs. Via this strategy, we have generated a yellow-green split-mNeonGreen2 1-10/11 that improves the ratio of complemented signal to the background of FP 1-10 -expressing cells compared to the commonly used split GFP 1-10/11 ; as well as a 10-fold brighter red-colored split-sfCherry2 1-10/11 . Based on split sfCherry2, we have engineered a photoactivatable variant that enables single-molecule localization-based super-resolution microscopy. We have demonstrated dual-color endogenous protein tagging with sfCherry2 11 and GFP 11 , revealing that endoplasmic reticulum translocon complex Sec61B has reduced abundance in certain peripheral tubules. These new split FPs not only offer multiple colors for imaging interaction networks of endogenous proteins, but also hold the potential to provide orthogonal handles for biochemical isolation of native protein complexes. 5,6 , identification of cell contacts and synapses 7,8 , as well as scaffolding protein assembly 3, 9, 10 . Recently, they have also enabled the generation of large-scale human cell line libraries with fluorescently tagged endogenous proteins through CRISPR/Cas9-based gene editing 11 .So far, the most commonly used self-complementing split FP was GFP 1-10D7/11M3 OPT (which we refers to as GFP 1-10/11 ), engineered from super-folder GFP (sfGFP) 12 . With the splitting point between the tenth and eleventh β-strands, the resulting GFP 11 fragment is a 16-amino acid (a.a.) short peptide. The corresponding GFP 1-10 fragment remains almost non-fluorescent until complementation, making GFP 1-10/11 well suited for protein labeling by fusing GFP 11 to the target protein and over-expressing GFP 1-10 in the corresponding subcellular compartments. However, there lacks a second, orthogonal split FP system with comparable complementation performance for multicolor imaging and multiplexed scaffolding of protein assembly. Previously, a sfCherry 1-10/11 system 3 was derived from super-folder Cherry, an mCherry variant optimized for folding efficiency 13 . However, its overall fluorescent brightness is substantially weaker than an intact sfCherry fusion, potentially due to its limited complementation efficiency 3 . Although two-color imaging with sfCherry 1-10/11 and GFP 1-10/11 has been done using tandem sfCherry 11 to amplify the sfCherry signal for over-expressed targets, it is still too dim to detect most endogenous proteins.In this paper, we report a screening strategy for the direct engineering of self-complementing split FPs. Using this strategy, we have generated a yellow-green-colored mNeonGreen2 1-10/11 (mNG2) that has an improved ratio of complemented signal to the background of FP 1-10 -expressing cells as compared to GFP 1-10/11 , as well as a red-colored sfCherry2 1-...

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Section: Discussionmentioning

confidence: 99%

Improved Split Fluorescent Proteins for Endogenous Protein Labeling

Feng

Sekine

Pessino

et al. 2017

Preprint

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“…To make these plasmids, the gfpmut2 gene was amplified by PCR from pMK021 18 using primers MKF013/MKR013, the mturquoise2 gene 51 was amplified by PCR from pDP428, a generous gift from Daniel B. Kearns and Ethan Garner using primers MKF011/MKR011 and the mko gene was amplified by PCR from pCN005 52 using primers MKF014/MKR014. The amplified genes were used to replace mkate2 by overlap extension PCR cloning.…”

Section: Methodsmentioning

confidence: 99%

Surface-attached molecules control Staphylococcus aureus quorum sensing and biofilm development

et al. 2017

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Bacteria use a process called quorum sensing to communicate and orchestrate collective behaviors including virulence factor secretion and biofilm formation. Quorum sensing relies on production, release, accumulation, and population-wide detection of signal molecules called autoinducers. Here, we develop concepts to coat surfaces with quorum-sensing-manipulation molecules as a method to control collective behaviors. We probe this strategy using Staphylococcus aureus. Pro- and anti-quorum-sensing molecules can be covalently attached to surfaces using click chemistry, where they retain their abilities to influence bacterial behaviors. We investigate key features of the compounds, linkers, and surfaces necessary to appropriately position molecules to interact with cognate receptors, and the ability of modified surfaces to resist long-term storage, repeated infections, host plasma components, and flow-generated stresses. Our studies highlight how this surface approach can be used to make colonization-resistant materials against S. aureus and other pathogens and how the approach can be adapted to promote beneficial behaviors of bacteria on surfaces.

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“…Since the cyan FPs (CFP) are optimally excited close to the power maximum of the titanium-sapphire lasers used in most TPLSM systems, we focused our evaluations of potential FRET donors on the newer variants of the CFPs that have improved brightness and photostability, and no photo-switching behavior [34][35][36][37] . Based on previous studies demonstrating that wavelengths near 800 nm could be used for relatively selective excitation of CFP over YFP 38,39 , we focused our evaluations of potential FRET acceptors on newer, improved variants of YFP.…”

Section: Development Of the Protocolmentioning

confidence: 99%

“…Increase illumination level. 29,36 Fluorescence image decreases in intensity and/or clarity over time.…”

Section: Measure Viral Titermentioning

confidence: 99%

A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy

2016

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Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%

Cited by 688 publications

References 36 publications

Improved Split Fluorescent Proteins for Endogenous Protein Labeling

Improved Split Fluorescent Proteins for Endogenous Protein Labeling

Surface-attached molecules control Staphylococcus aureus quorum sensing and biofilm development

A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy

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