scholarly journals Switching between Ultrafast Pathways Enables a Green-Red Emission Ratiometric Fluorescent-Protein-Based Ca2+ Biosensor

2021 ◽  
Vol 22 (1) ◽  
pp. 445
Author(s):  
Longteng Tang ◽  
Shuce Zhang ◽  
Yufeng Zhao ◽  
Nikita D. Rozanov ◽  
Liangdong Zhu ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Dynamic Range ◽  
Life Sciences ◽  
Green Emission ◽  
Red Fluorescent Protein ◽  
Working Mechanism ◽  
Dark State ◽  
Short Hydrogen Bond ◽  
Ratio Change ◽  
First Time

Ratiometric indicators with long emission wavelengths are highly preferred in modern bioimaging and life sciences. Herein, we elucidated the working mechanism of a standalone red fluorescent protein (FP)-based Ca2+ biosensor, REX-GECO1, using a series of spectroscopic and computational methods. Upon 480 nm photoexcitation, the Ca2+-free biosensor chromophore becomes trapped in an excited dark state. Binding with Ca2+ switches the route to ultrafast excited-state proton transfer through a short hydrogen bond to an adjacent Glu80 residue, which is key for the biosensor’s functionality. Inspired by the 2D-fluorescence map, REX-GECO1 for Ca2+ imaging in the ionomycin-treated human HeLa cells was achieved for the first time with a red/green emission ratio change (ΔR/R0) of ~300%, outperforming many FRET- and single FP-based indicators. These spectroscopy-driven discoveries enable targeted design for the next-generation biosensors with larger dynamic range and longer emission wavelengths.

scholarly journals Red Fluorescent Genetically Encoded Voltage Indicators with Millisecond Responsiveness

Sensors ◽  
2019 ◽  
Vol 19 (13) ◽  
pp. 2982 ◽  
Author(s):  
Liubov A. Kost ◽  
Violetta O. Ivanova ◽  
Pavel M. Balaban ◽  
Konstantin A. Lukyanov ◽  
Evgeny S. Nikitin ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Dynamic Range ◽  
Response Speed ◽  
Red Fluorescent Protein ◽  
Positive Slope ◽  
Reporter Protein ◽  
Fluorescent Indicators ◽  
Linker Length ◽  
Final Performance ◽  
Voltage Indicator

Genetically encoded fluorescent indicators typically consist of the sensitive and reporter protein domains connected with the amino acid linkers. The final performance of a particular indicator may depend on the linker length and composition as strong as it depends on the both domains nature. Here we aimed to optimize interdomain linkers in VSD-FR189-188—a recently described red fluorescent protein-based voltage indicator. We have tested 13 shortened linker versions and monitored the dynamic range, response speed and polarity of the corresponding voltage indicator variants. While the new indicators didn’t show a contrast enhancement, some of them carrying very short interdomain linkers responded 25-fold faster than the parental VSD-FR189-188. Also we found the critical linker length at which fluorescence response to voltage shift changes its polarity from negative to positive slope. Our observations thus make an important contribution to the designing principles of the fluorescent protein-derived voltage indicators.

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

scholarly journals Novel Regeneration Approach for Creating Reusable FO-SPR Probes with NTA Surface Chemistry

Nanomaterials ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 186
Author(s):  
Jia-Huan Qu ◽  
Karen Leirs ◽  
Remei Escudero ◽  
Žiga Strmšek ◽  
Roman Jerala ◽  
...  
Keyword(s):  
Surface Chemistry ◽  
Fluorescent Protein ◽  
Cost Effective ◽  
Nitrilotriacetic Acid ◽  
Antibody Fragments ◽  
Rapid Screening ◽  
Red Fluorescent Protein ◽  
Effective Manner ◽  
Surface Regeneration ◽  
Sensitivity Specificity

To date, surface plasmon resonance (SPR) biosensors have been exploited in numerous different contexts while continuously pushing boundaries in terms of improved sensitivity, specificity, portability and reusability. The latter has attracted attention as a viable alternative to disposable biosensors, also offering prospects for rapid screening of biomolecules or biomolecular interactions. In this context here, we developed an approach to successfully regenerate a fiber-optic (FO)-SPR surface when utilizing cobalt (II)-nitrilotriacetic acid (NTA) surface chemistry. To achieve this, we tested multiple regeneration conditions that can disrupt the NTA chelate on a surface fully saturated with His6-tagged antibody fragments (scFv-33H1F7) over ten regeneration cycles. The best surface regeneration was obtained when combining 100 mM EDTA, 500 mM imidazole and 0.5% SDS at pH 8.0 for 1 min with shaking at 150 rpm followed by washing with 0.5 M NaOH for 3 min. The true versatility of the established approach was proven by regenerating the NTA surface for ten cycles with three other model system bioreceptors, different in their size and structure: His6-tagged SARS-CoV-2 spike fragment (receptor binding domain, RBD), a red fluorescent protein (RFP) and protein origami carrying 4 RFPs (Tet12SN-RRRR). Enabling the removal of His6-tagged bioreceptors from NTA surfaces in a fast and cost-effective manner can have broad applications, spanning from the development of biosensors and various biopharmaceutical analyses to the synthesis of novel biomaterials.

A brilliant monomeric red fluorescent protein to visualize cytoskeleton dynamics inDictyostelium

FEBS Letters ◽  
2004 ◽  
Vol 577 (1-2) ◽  
pp. 227-232 ◽  
Author(s):  
Markus Fischer ◽  
Ilka Haase ◽  
Evelyn Simmeth ◽  
Günther Gerisch ◽  
Annette Müller-Taubenberger
Keyword(s):  
Fluorescent Protein ◽  
Red Fluorescent Protein ◽  
Cytoskeleton Dynamics

scholarly journals Unique interactions between the chromophore and glutamate 16 lead to far-red emission in a red fluorescent protein

2009 ◽  
Vol 18 (2) ◽  
pp. 460-466 ◽  
Author(s):  
Xiaokun Shu ◽  
Lei Wang ◽  
Leslie Colip ◽  
Karen Kallio ◽  
S. James Remington
Keyword(s):  
Fluorescent Protein ◽  
Red Fluorescent Protein ◽  
Red Emission
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