Extending fluorescence microscopy into anaerobic environments

Chia, Hannah E; Marsh, E. Neil G.; Biteen, Julie S.

doi:10.1016/j.cbpa.2019.05.008

Cited by 49 publications

(59 citation statements)

References 62 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The N-terminal 37 amino acids of FRDg that include the Dxx(s/t)(s/g)AS consensus motif [32] are sufficient as substrate for efficient flavinylation when fused to different proteins localised in the cytoplasm or in the glycosome. We also confirmed that Ser9 in this motif is essential for flavinylation [64], in agreement with Serebryakova et al [68,69], a broad functional pH range (4)(5)(6)(7)(8)(9)(10)(11) [70] and their small size -for iLOV approximately 10 kDa [71]. Other variants, such as miniSOG, can generate reactive singlet oxygen upon illumination, which is exploited for electron microscopy [72].…”

Section: Pyrrhocorissupporting

confidence: 90%

Efficient flavinylation of glycosomal fumarate reductase by its own ApbE domain in Trypanosoma brucei

Schenk

Bachmaier

Bringaud

et al. 2020

Preprint

View full text Add to dashboard Cite

A subset of flavoproteins has a covalently attached flavin prosthetic group enzymatically attached via phosphoester bonding. In prokaryotes, this is catalysed by ApbE flavin transferases. ApbE-like domains are present in few eukaryotic taxa, e.g. the N-terminal domain of fumarate reductase (FRD) of Trypanosoma, a parasitic protist known as a tropical pathogen causing African sleeping sickness. We use the versatile reverse genetic tools available for Trypanosoma to investigate the flavinylation of glycosomal FRD (FRDg) in vivo in the physiological and organellar context. Using direct in-gel fluorescence detection of covalently attached flavin as proxy for activity, we show that the ApbE-like domain of FRDg has flavin transferase activity in vivo. The ApbE domain is preceded by a consensus flavinylation target motif at the extreme N-terminus of FRDg, and serine 9 in this motif is essential as flavin acceptor. The preferred mode of flavinylation in the glycosome was addressed by stoichiometric expression and comparison of native and catalytically dead ApbE domains. In addition to the trans-flavinylation activity, the ApbE domain catalyses the intramolecular cis-flavinylation with at least 5-fold higher efficiency. We discuss how the higher efficiency due to unusual fusion of the ApbE domain to its substrate protein FRD may provide a selective advantage by faster FRD biogenesis during rapid metabolic adaptation of trypanosomes. The first 37 amino acids of FRDg, including the consensus motif, are sufficient as flavinylation target upon fusion to other proteins. We propose FRDg(1-37) as 4 kDa heat-stable, detergent-resistant fluorescent protein tag and suggest its use as a new tool to study glycosomal protein import.

show abstract

Section: Pyrrhocorissupporting

confidence: 90%

Efficient flavinylation of glycosomal fumarate reductase by its own ApbE domain in Trypanosoma brucei

Schenk

Bachmaier

Bringaud

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…Next to the comparably small shift in the spectra, we noticed a strong reduction in fluorescent intensity in the two iLOV mutants compared to iLOV (Figure 3). This is particularly problematic due to low brightness of iLOV itself and FbFPs in general 7,12 . The fluorescent intensity of iLOV L470T/ Q489K was reduced around 10-fold, the intensity of iLOV iLOV V392K/F410V/A426S even around 100-fold compared to WT iLOV.…”

Section: Resultsmentioning

confidence: 99%

“…Several rounds of DNA shuffling led to a mutation in a key cysteine residue and triggering the emission of fluorescence 7 . So far, all experimentally obtained FbFPs share spectral features similar to GFP 12 . However, FbFPs possessed relatively weak fluorescent intensity until very recently 13,14 .…”

Section: Introductionmentioning

confidence: 83%

Experimental characterization ofin silicored-shift predicted iLOV^L470T/Q489Kand iLOV^{V392K/F410V/A426S}mutants

Wehler

Öztürk

2021

Preprint

View full text Add to dashboard Cite

iLOV is a flavin mononucleotide (FMN)-binding fluorescent protein (FbFP) with excitation and emission spectra similar to those of the green fluorescent protein (GFP). Importantly, contrary to GFP, iLOV fluoresces independently of molecular oxygen, making its usage in low-oxygen conditions possible. Moreover, iLOV is smaller than GFP, increasing the likelihood of retaining full functionality when creating fusions with proteins of interest. Nonetheless, GFP remains to date the most widely used FP in molecular biology, also due to the availability of multiple spectrally tuned variants allowing multi-color imaging experiments. To expand the range of applications of iLOV, spectrally tuned red-shift variants are desirable to have reduced phototoxicity and better tissue penetration. Here we experimentally tested two iLOV mutants, iLOV L470T/Q489K and iLOV V392K/F410V/A426S, which were previously computationally proposed to have red-shifted excitation and emission spectra. We found that only the triple mutant has moderately red-shifted excitation and emission spectra. Both mutants exhibit strongly decreased brightness, which impedes their employment in live cell imaging. Finally, we show that the single V392K mutation suffices to red-shift the emission spectrum as well as abolishes fluorescence of iLOV.

show abstract

“…Flavin-based fluorescent proteins (FbFPs) are derived from light-oxygen-voltage (LOV) sensing domains, utilised by plants and bacteria as blue-light photoreceptors [41,42]. Their application as fluorescent reporters offers several advantages over GFP-like fluorescent proteins, such as oxygen-independent fluorescence [43,44], a broad functional pH range (4-11) [45] and their small sizefor iLOV~10 kDa [46]. Other variants, such as miniSOG, can generate reactive singlet oxygen upon illumination, which is exploited for electron microscopy [47].…”

Section: Discussionmentioning

confidence: 99%

Efficient flavinylation of glycosomal fumarate reductase by its own ApbE domain in Trypanosoma brucei

et al. 2021

View full text Add to dashboard Cite

A subset of flavoproteins has a covalently attached flavin prosthetic group enzymatically attached via phosphoester bonding. In prokaryotes, this is catalysed by alternative pyrimidine biosynthesis E (ApbE) flavin transferases. ApbE-like domains are present in few eukaryotic taxa, for example the N-terminal domain of fumarate reductase (FRD) of Trypanosoma, a parasitic protist known as a tropical pathogen causing African sleeping sickness. We use the versatile reverse genetic tools available for Trypanosoma to investigate the flavinylation of glycosomal FRD (FRDg) in vivo in the physiological and organellar context. Using direct in-gel fluorescence detection of covalently attached flavin as proxy for activity, we show that the ApbE-like domain of FRDg has flavin transferase activity in vivo. The ApbE domain is preceded by a consensus flavinylation target motif at the extreme N terminus of FRDg, and serine 9 in this motif is essential as flavin acceptor. The preferred mode of flavinylation in the glycosome was addressed by stoichiometric expression and comparison of native and catalytically inactive ApbE domains. In addition to the trans-flavinylation activity, the ApbE domain catalyses the intramolecular cis-flavinylation with at least fivefold higher efficiency. We discuss how the higher efficiency due to unusual fusion of the ApbE domain to its substrate protein FRD may provide a selective advantage by faster FRD biogenesis during rapid metabolic adaptation of trypanosomes. The first 37 amino acids of FRDg, including the consensus motif, are sufficient as flavinylation target upon fusion to other proteins. We propose FRDg(1-37) as 4-kDa heat-stable, detergent-resistant fluorescent protein tag and suggest its use as a new tool to study glycosomal protein import.

show abstract

Extending fluorescence microscopy into anaerobic environments

Cited by 49 publications

References 62 publications

Efficient flavinylation of glycosomal fumarate reductase by its own ApbE domain in Trypanosoma brucei

Efficient flavinylation of glycosomal fumarate reductase by its own ApbE domain in Trypanosoma brucei

Experimental characterization ofin silicored-shift predicted iLOV^L470T/Q489Kand iLOV^{V392K/F410V/A426S}mutants

Efficient flavinylation of glycosomal fumarate reductase by its own ApbE domain in Trypanosoma brucei

Contact Info

Product

Resources

About

Extending fluorescence microscopy into anaerobic environments

Cited by 49 publications

References 62 publications

Efficient flavinylation of glycosomal fumarate reductase by its own ApbE domain in Trypanosoma brucei

Efficient flavinylation of glycosomal fumarate reductase by its own ApbE domain in Trypanosoma brucei

Experimental characterization ofin silicored-shift predicted iLOVL470T/Q489Kand iLOVV392K/F410V/A426Smutants

Efficient flavinylation of glycosomal fumarate reductase by its own ApbE domain in Trypanosoma brucei

Contact Info

Product

Resources

About

Experimental characterization ofin silicored-shift predicted iLOV^L470T/Q489Kand iLOV^{V392K/F410V/A426S}mutants