scholarly journals A New Fluorogenic Substrate for Granzyme B Based on Fluorescence Resonance Energy Transfer

2020 ◽  
Vol 3 (1) ◽  
pp. 6
Author(s):  
Cátia D. F. Martins ◽  
M. Manuela M. Raposo ◽  
Susana P. G. Costa
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Solid Phase ◽  
Granzyme B ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Preparative Hplc ◽  
Fluorogenic Substrate ◽  
Acceptor Pair ◽  
Fluorescence Resonance

The synthesis and characterization of a new fluorogenic substrate for granzyme B (GzmB) is reported. The substrate design was based on the fluorescence resonance energy transfer (FRET) principle using 5-(2′-aminoethyl)aminonaphthalene sulfonic acid (Edans) and 4-[[4′-(N,N-dimethylamino)phenyl]diazenyl]benzoic acid (Dabcyl) as a donor–acceptor pair, linked to a specific sequence for GzmB (AAD), with an additional amino acid as the anchoring point (K). The tetrapeptide was synthesized by microwave-assisted solid-phase peptide synthesis (MW-SPPS) and coupled to Dabcyl and Edans at its N- and C-termini, respectively. The obtained probe was purified by semi-preparative HPLC and characterized by NMR, UV/Vis absorption and fluorescence spectroscopy and mass spectrometry.

scholarly journals Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer

2004 ◽  
Vol 381 (1) ◽  
pp. 307-312 ◽  
Author(s):  
Satoshi KARASAWA ◽  
Toshio ARAKI ◽  
Takeharu NAGAI ◽  
Hideaki MIZUNO ◽  
Atsushi MIYAWAKI
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Acceptor Pair ◽  
Donor And Acceptor ◽  
Donor Acceptor ◽  
Donor Acceptor Pair ◽  
Fluorescence Resonance

GFP (green fluorescent protein)-based FRET (fluorescence resonance energy transfer) technology has facilitated the exploration of the spatio-temporal patterns of cellular signalling. While most studies have used cyan- and yellow-emitting FPs (fluorescent proteins) as FRET donors and acceptors respectively, this pair of proteins suffers from problems of pH-sensitivity and bleeding between channels. In the present paper, we demonstrate the use of an alternative additional donor/acceptor pair. We have cloned two genes encoding FPs from stony corals. We isolated a cyan-emitting FP from Acropara sp., whose tentacles exhibit cyan coloration. Similar to GFP from Renilla reniformis, the cyan FP forms a tight dimeric complex. We also discovered an orange-emitting FP from Fungia concinna. As the orange FP exists in a complex oligomeric structure, we converted this protein into a monomeric form through the introduction of three amino acid substitutions, recently reported to be effective for converting DsRed into a monomer (Clontech). We used the cyan FP and monomeric orange FP as a donor/acceptor pair to monitor the activity of caspase 3 during apoptosis. Due to the close spectral overlap of the donor emission and acceptor absorption (a large Förster distance), substantial pH-resistance of the donor fluorescence quantum yield and the acceptor absorbance, as well as good separation of the donor and acceptor signals, the new pair can be used for more effective quantitative FRET imaging.

scholarly journals Highly efficient fluorescence resonance energy transfer in co-encapsulated BODIPY nanoparticles

2021 ◽  
Vol 12 (4) ◽  
pp. 361-367
Author(s):  
Priyadarshine Hewavitharanage ◽  
Launa Steele ◽  
Isaac Dickenson
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Acceptor Pair ◽  
Highly Efficient ◽  
Fluorescence Measurements ◽  
Donor Acceptor ◽  
Donor Acceptor Pair ◽  
Fluorescence Resonance

Fluorescence resonance energy transfer (FRET) is a powerful tool used in a wide range of applications due to its high sensitivity and many other advantages. Co-encapsulation of a donor and an acceptor in nanoparticles is a useful strategy to bring the donor-acceptor pair in proximity for FRET. A highly efficient FRET system based on BODIPY-BODIPY (BODIPY:  boron-dipyrromethene) donor-acceptor pair in nanoparticles was synthesized. Nanoparticles were formed by co-encapsulating a green emitting BODIPY derivative (FRET donor, lmax = 501 nm) and a red emitting BODIPY derivative (FRET acceptor, lmax = 601 nm) in an amphiphilic polymer using the precipitation method. Fluorescence measurements of encapsulated BODIPY in water following 501 nm excitation caused a 3.6 fold enhancement of the acceptor BODIPY emission at 601 nm indicating efficient energy transfer between the green emitting donor BODIPY and the red emitting BODIPY acceptor with a 100 nm Stokes shift. The calculated FRET efficiency was 96.5%. Encapsulated BODIPY derivatives were highly stable under our experimental conditions.

Fluorescence resonance energy transfer between coumarin-derived mitochondrial F1-ATPase γ subunit and pyrenylmaleimide-labelled fragments of IF1 and c subunit

2002 ◽  
Vol 362 (2) ◽  
pp. 165-171 ◽  
Author(s):  
Alessandra BARACCA ◽  
Silvia BAROGI ◽  
Sara PAOLINI ◽  
Giorgio LENAZ ◽  
Giancarlo SOLAINI
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Structural Information ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Acceptor Pair ◽  
F1 Atpase ◽  
Γ Subunit ◽  
C Subunit ◽  
Fluorescence Resonance

We introduced a reporting group into a critical position of the mitochondrial F1-ATPase in order to gain structural information about enzyme—ligand complexes. Incubation of 7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM) with bovine heart mitochondrial F1-ATPase pretreated with 1mM sodium arsenite modified the only cysteine residue in the γ subunit (γ-Cys78), resulting in an enzyme—CPM fluorescent complex (CPM—F1) with an ATPase activity similar to that of the native enzyme. Transferred fluorescence of F1-bound CPM occurred when different peptide fragments of naturally binding polypeptides carrying a pyrenylmaleimide (PM) moiety were bound to the enzyme. Fluorescence resonance energy transfer (RET) from PM bound to cysteine residues associated with Glu40, Lys47 and Lys58 of fragments of the inhibitor protein (IF1) with CPM—F1 occurred with an efficiency of approx. 20, 21 and 3% respectively. The distance at which the efficiency of energy transfer was 50%, R0, for the CPM and PM donor/acceptor pair was 4.1nm, indicating that the three IF1 fragments must be within 6.7nm of γ-Cys78. RET from the PM-bound hydrophilic fragment of c subunit (residues 37–42) of the F1F0-ATPase complex and CPM-bound γ-Cys78 occurred with an efficiency of approx. 30%, indicating a distance of 4.7nm between the two fluorophores. Based on previous observations and on the present RET measurements, the hydrophilic loop of c subunit was located at the base of the F1 foot, and the N-terminal region of IF1 was located on the surface of F1 in the lower part of the α3β3 hexamer ring.

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