scholarly journals An endogenous green fluorescent protein–photoprotein pair in Clytia hemisphaerica eggs shows co-targeting to mitochondria and efficient bioluminescence energy transfer

Open Biology ◽  
2014 ◽  
Vol 4 (4) ◽  
pp. 130206 ◽  
Author(s):  
Cécile Fourrage ◽  
Karl Swann ◽  
Jose Raul Gonzalez Garcia ◽  
Anthony K. Campbell ◽  
Evelyn Houliston
Keyword(s):  
Energy Transfer ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Green Light ◽  
Resonance Energy ◽  
Green Fluorescent Proteins ◽  
Parallel Acquisition ◽  
Green Fluorescent

Green fluorescent proteins (GFPs) and calcium-activated photoproteins of the aequorin/clytin family, now widely used as research tools, were originally isolated from the hydrozoan jellyfish Aequora victoria . It is known that bioluminescence resonance energy transfer (BRET) is possible between these proteins to generate flashes of green light, but the native function and significance of this phenomenon is unclear. Using the hydrozoan Clytia hemisphaerica , we characterized differential expression of three clytin and four GFP genes in distinct tissues at larva, medusa and polyp stages, corresponding to the major in vivo sites of bioluminescence (medusa tentacles and eggs) and fluorescence (these sites plus medusa manubrium, gonad and larval ectoderms). Potential physiological functions at these sites include UV protection of stem cells for fluorescence alone, and prey attraction and camouflaging counter-illumination for bioluminescence. Remarkably, the clytin2 and GFP2 proteins, co-expressed in eggs, show particularly efficient BRET and co-localize to mitochondria, owing to parallel acquisition by the two genes of mitochondrial targeting sequences during hydrozoan evolution. Overall, our results indicate that endogenous GFPs and photoproteins can play diverse roles even within one species and provide a striking and novel example of protein coevolution, which could have facilitated efficient or brighter BRET flashes through mitochondrial compartmentalization.

scholarly journals Intramolecular Fluorescence Resonance Energy Transfer between Fused Autofluorescent Proteins Reveals Rearrangements of the N- and C-terminal Segments of the Plasma Membrane Ca2+ Pump Involved in the Activation

2007 ◽  
Vol 282 (49) ◽  
pp. 35440-35448 ◽  
Author(s):  
Gerardo R. Corradi ◽  
Hugo P. Adamo
Keyword(s):  
Plasma Membrane ◽  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Fluorescent Proteins ◽  
Average Distance ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Green Fluorescent Proteins ◽  
Fluorescence Resonance ◽  
Green Fluorescent

The blue and green fluorescent proteins (BFP and GFP) have been fused at the N- and C-terminal ends, respectively, of the plasma membrane Ca2+ pump (PMCA) isoform 4xb (hPMCA4xb). The fusion protein was successfully expressed in yeast and purified by calmodulin affinity chromatography. Despite the presence of the fused autofluorescent proteins BFP-PMCA-GFP performed similarly to the wild-type enzyme with respect to Ca2+-ATPase activity and sensitivity to calmodulin activation. In the autoinhibited state BFP-PMCA-GFP exhibited a significant intramolecular fluorescence resonance energy transfer (FRET) consistent with the location of the fluorophores at an average distance of 45Å. The FRET intensity in BFP-PMCA-GFP decreased when the enzyme was activated either by Ca2+-calmodulin, partial proteolysis, or acidic lipids. Moreover, FRET decreased and became insensitive to calmodulin when hPMCA4xb was activated by mutation D170N in BFP-PMCA(D170N)-GFP. The results suggest that the ends of the PMCA are in close proximity in the autoinhibited conformation, and they separate or reorient when the PMCA achieves its final activated conformation.

Molecular probes: insights into design and analysis from computational and physical chemistry

2008 ◽  
Vol 36 (1) ◽  
pp. 46-50 ◽  
Author(s):  
Felicity L. Mitchell ◽  
Gabriel E. Marks ◽  
Elena V. Bichenkova ◽  
Kenneth T. Douglas ◽  
Richard A. Bryce
Keyword(s):  
Physical Chemistry ◽  
Energy Transfer ◽  
Experimental Approach ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Molecular Probes ◽  
Green Fluorescent ◽  
Nanoscale Level

The application of new molecular diagnostics to probe cellular process in vivo is leading to a greater understanding of molecular cytology at a sub-nanoscale level and is opening the way to individualized medicines. We review here three distinct fluorescence-based molecular probes, HyBeacons™, split-probe exciplexes and GFP (green fluorescent protein)-based FRET (fluorescence resonance energy transfer) systems. Through this, we highlight the insights into the mechanism and design that a combined computational and experimental approach can yield.

Fluorescent Indicators for Calcium Based on Green Fluorescent Proteins (GFPs) and Calmodulin

1998 ◽  
Vol 4 (S2) ◽  
pp. 446-447
Author(s):  
A. Miyawaki ◽  
J. Llopis ◽  
R. Heim ◽  
J.M. McCafFery ◽  
J.A. Adams ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Green Fluorescent Proteins ◽  
Fluorescent Indicators ◽  
Calmodulin Binding ◽  
Green Fluorescent ◽  
Partner Proteins

Cytosolic and organellar free Ca2+ concentrations are very dynamic; they are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations.Green fluorescent protein (GFP) is a spontaneously fluorescent protein from the jelly fish Aequorea victoria. Its cDNA can be concatenated with those encoding many other proteins, and the resulting fusion proteins are usually fluorescent and often preserve the biochemical functions and cellular localizations of the partner proteins. Mutagenesis has produced GFP mutants with shifted wavelengths of excitation or emission that can serve as donors and acceptors for fluorescence resonance energy transfer (FRET).Our new indicators consist of tandem fusions of a blue- or cyan-emitting mutant of GFP, calmodulin (CaM), the calmodulin-binding peptide Ml3, and an enhanced green- or yellow-emitting GFP. Binding of Ca2+ makes calmodulin wrap around the Ml3 domain, increasing the FRET between the flanking GFPs (Fig. 1).

scholarly journals The effect of proximity on the function and energy transfer capability of fluorescent protein pairs

2019 ◽  
Author(s):  
Jacob R. Pope ◽  
Rachel L. Johnson ◽  
W. David Jamieson ◽  
Harley L Worthy ◽  
Senthilkumar D. Kailasam ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Protein Interactions ◽  
Fluorescent Properties ◽  
Transition Dipole ◽  
Green Fluorescent

AbstractFluorescent proteins (FPs) are commonly used in pairs to monitor dynamic biomolecular events through changes in their proximity via distance dependent processes such as Förster resonance energy transfer (FRET). Many FPs have a tendency to oligomerise, which is likely to be promoted through attachment to associating proteins through increases in local FP concentration. We show here that on association of FP pairs, the inherent function of the FPs can alter. Artificial dimers were constructed using a bioorthogonal Click chemistry approach that combined a commonly used green fluorescent protein (superfolder GFP) with itself, a yellow FP (Venus) or a red FP (mCherry). In each case dimerisation changes the inherent fluorescent properties, including FRET capability. The GFP homodimer demonstrated synergistic behaviour with the dimer being brighter than the sum of the two monomers. The structure of the GFP homodimer revealed that a water-rich interface is formed between the two monomers, with the chromophores being in close proximity with favourable transition dipole alignments. Dimerisation of GFP with Venus results in a complex displaying ∼86% FRET efficiency, which is significantly below the near 100% efficiency predicted. When GFP is complexed with mCherry, FRET and mCherry fluorescence itself is essentially lost. Thus, the simple assumptions used when monitoring interactions between proteins via FP FRET may not always hold true, especially under conditions whereby the protein-protein interactions promote FP interaction.Abstract Figure

scholarly journals Far-red fluorescent tag for protein labelling

2002 ◽  
Vol 368 (1) ◽  
pp. 17-21 ◽  
Author(s):  
Arkady F. FRADKOV ◽  
Vladislav V. VERKHUSHA ◽  
Dmitry B. STAROVEROV ◽  
Maria E. BULINA ◽  
Yurii G. YANUSHEVICH ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Sea Anemones ◽  
Practical Applications ◽  
In Vivo Labelling ◽  
Covalently Linked ◽  
Green Fluorescent

Practical applications of green fluorescent protein ('GFP')-like fluorescent proteins (FPs) from species of the class Anthozoa (sea anemones, corals and sea pens) are strongly restricted owing to their oligomeric nature. Here we suggest a strategy to overcome this problem by the use of two covalently linked identical red FPs as non-oligomerizing fusion tags. We have applied this approach to the dimeric far-red fluorescent protein HcRed1 and have demonstrated superiority of the tandem tag in the in vivo labelling of fine cytoskeletal structures and tiny nucleoli. In addition, a possibility of effective fluorescence resonance energy transfer ('FRET') between enhanced yellow FP mutant ('EYFP') and tandem HcRed1 was demonstrated in a protease assay.

scholarly journals Measuring ligand-dependent and ligand-independent interactions between nuclear receptors and associated proteins using Bioluminescence Resonance Energy Transfer (BRET2)

2006 ◽  
Vol 4 (1) ◽  
pp. nrs.04021 ◽  
Author(s):  
Kristen L. Koterba ◽  
Brian G. Rowan
Keyword(s):  
Energy Transfer ◽  
Protein Interactions ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Renilla Luciferase ◽  
Receptor Protein ◽  
Protein Protein Interactions

Bioluminescent resonance energy transfer (BRET2) is a recently developed technology for the measurement of protein-protein interactions in a live, cell-based system. BRET2 is characterized by the efficient transfer of excited energy between a bioluminescent donor molecule (Renilla luciferase) and a fluorescent acceptor molecule (a mutant of Green Fluorescent Protein (GFP2)). The BRET2 assay offers advantages over fluorescence resonance energy transfer (FRET) because it does not require an external light source thereby eliminating problems of photobleaching and autoflourescence. The absence of contamination by light results in low background that permits detection of very small changes in the BRET2 signal. BRET2 is dependent on the orientation and distance between two fusion proteins and therefore requires extensive preliminary standardization experiments to conclude a positive BRET2 signal independent of variations in protein titrations and arrangement in tertiary structures. Estrogen receptor (ER) signaling is modulated by steroid receptor coactivator 1 (SRC-1). To establish BRET2 in a ligand inducible system we used SRC-1 as the donor moiety and ER as the acceptor moiety. Expression and functionality of the fusion proteins were assessed by transient transfection in HEK-293 cells followed by Western blot analysis and measurement of ER-dependent reporter gene activity. These preliminary determinations are required prior to measuring nuclear receptor protein-protein interactions by BRET2. This article describes in detail the BRET2 methodology for measuring interaction between full-length ER and coregulator proteins in real-time, in an in vivo environment.

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