scholarly journals An enhanced molecular tension sensor based on bioluminescence resonance energy transfer (BRET)

2019 ◽  
Author(s):  
Eric J. Aird ◽  
Kassidy J. Tompkins ◽  
Wendy R. Gordon
Keyword(s):  
Energy Transfer ◽  
Dynamic Range ◽  
Resonance Energy Transfer ◽  
Focal Adhesions ◽  
Resonance Energy ◽  
Cellular Protein ◽  
Bioluminescence Resonance Energy Transfer ◽  
Tension Sensor ◽  
Focal Adhesion Protein

ABSTRACTMolecular tension sensors measure piconewton forces experienced by individual proteins in the context of the cellular microenvironment. Current genetically-encoded tension sensors use FRET to report on extension of an elastic peptide encoded in a cellular protein of interest. Here we present the development and characterization of a new type of molecular tension sensor based on bioluminescence resonance energy transfer (BRET) which exhibits more desirable spectral properties and an enhanced dynamic range compared to other molecular tension sensors. Moreover, it avoids many disadvantages of FRET measurements in cells, including heating of the sample, autofluorescence, photobleaching, and corrections of direct acceptor excitation. We benchmark the sensor by inserting it into the canonical mechanosensing focal adhesion protein vinculin, observing highly resolved gradients of tensional changes across focal adhesions. We anticipate that the BRET-TS will expand the toolkit available to study mechanotransduction at a molecular level and allow potential extension to an in vivo context.

scholarly journals Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach

2007 ◽  
Vol 407 (1) ◽  
pp. 69-77 ◽  
Author(s):  
Michael Russwurm ◽  
Florian Mullershausen ◽  
Andreas Friebe ◽  
Ronald Jäger ◽  
Corina Russwurm ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Systematic Approach ◽  
Dynamic Range ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Binding Domains ◽  
Fluorescence Resonance

The intracellular signalling molecule cGMP regulates a variety of physiological processes, and so the ability to monitor cGMP dynamics in living cells is highly desirable. Here, we report a systematic approach to create FRET (fluorescence resonance energy transfer)-based cGMP indicators from two known types of cGMP-binding domains which are found in cGMP-dependent protein kinase and phosphodiesterase 5, cNMP-BD [cyclic nucleotide monophosphate-binding domain and GAF [cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA] respectively. Interestingly, only cGMP-binding domains arranged in tandem configuration as in their parent proteins were cGMP-responsive. However, the GAF-derived sensors were unable to be used to study cGMP dynamics because of slow response kinetics to cGMP. Out of 24 cGMP-responsive constructs derived from cNMP-BDs, three were selected to cover a range of cGMP affinities with an EC50 between 500 nM and 6 μM. These indicators possess excellent specifity for cGMP, fast binding kinetics and twice the dynamic range of existing cGMP sensors. The in vivo performance of these new indicators is demonstrated in living cells and validated by comparison with cGMP dynamics as measured by radioimmunoassays.

scholarly journals Quantum dot–NanoLuc bioluminescence resonance energy transfer enables tumor imaging and lymph node mapping in vivo

2016 ◽  
Vol 52 (43) ◽  
pp. 6997-7000 ◽  
Author(s):  
Anyanee Kamkaew ◽  
Haiyan Sun ◽  
Christopher G. England ◽  
Liang Cheng ◽  
Zhuang Liu ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Lymph Node ◽  
Quantum Dot ◽  
Tumor Imaging ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bioluminescence Resonance Energy Transfer ◽  
Lymph Node Mapping

A small luciferase (Nluc) was conjugated to QDs as a bioluminescence resonance energy transfer (BRET) pair.

Development of a Bioluminescence Resonance Energy-Transfer Assay for Estrogen-Like Compound in Vivo Monitoring

2004 ◽  
Vol 76 (23) ◽  
pp. 7069-7076 ◽  
Author(s):  
Elisa Michelini ◽  
Mara Mirasoli ◽  
Matti Karp ◽  
Marko Virta ◽  
Aldo Roda
Keyword(s):  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bioluminescence Resonance Energy Transfer ◽  
In Vivo Monitoring

Engineering with NanoLuc: a playground for the development of bioluminescent protein switches and sensors

2020 ◽  
Vol 48 (6) ◽  
pp. 2643-2655
Author(s):  
Lieuwe Biewenga ◽  
Bas J.H.M. Rosier ◽  
Maarten Merkx
Keyword(s):  
Energy Transfer ◽  
Cell Biology ◽  
Chemical Biology ◽  
Dynamic Range ◽  
State Of The Art ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bioluminescence Resonance Energy Transfer ◽  
Advantages And Disadvantages ◽  
Bioluminescent Protein

The small engineered luciferase NanoLuc has rapidly become a powerful tool in the fields of biochemistry, chemical biology, and cell biology due to its exceptional brightness and stability. The continuously expanding NanoLuc toolbox has been employed in applications ranging from biosensors to molecular and cellular imaging, and currently includes split complementation variants, engineering techniques for spectral tuning, and bioluminescence resonance energy transfer-based concepts. In this review, we provide an overview of state-of-the-art NanoLuc-based sensors and switches with a focus on the underlying protein engineering approaches. We discuss the advantages and disadvantages of various strategies with respect to sensor sensitivity, modularity, and dynamic range of the sensor and provide a perspective on future strategies and applications.

scholarly journals Self-illuminating in vivo lymphatic imaging using a bioluminescence resonance energy transfer quantum dot nano-particle

2010 ◽  
Vol 6 (1) ◽  
pp. 55-59 ◽  
Author(s):  
Nobuyuki Kosaka ◽  
Makoto Mitsunaga ◽  
Sukanta Bhattacharyya ◽  
Steven C. Miller ◽  
Peter L. Choyke ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Quantum Dot ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Nano Particle ◽  
Bioluminescence Resonance Energy Transfer ◽  
Lymphatic Imaging

scholarly journals Integration of Nanomaterials and Bioluminescence Resonance Energy Transfer Techniques for Sensing Biomolecules

Biosensors ◽  
2019 ◽  
Vol 9 (1) ◽  
pp. 42 ◽  
Author(s):  
Eugene Hwang ◽  
Jisu Song ◽  
Jin Zhang
Keyword(s):  
Quantum Dots ◽  
Energy Transfer ◽  
Uv Light ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
External Electromagnetic Field ◽  
Ease Of Use ◽  
Radiative Energy ◽  
Bioluminescence Resonance Energy Transfer

Bioluminescence resonance energy transfer (BRET) techniques offer a high degree of sensitivity, reliability and ease of use for their application to sensing biomolecules. BRET is a distance dependent, non-radiative energy transfer, which uses a bioluminescent protein to excite an acceptor through the resonance energy transfer. A BRET sensor can quickly detect the change of a target biomolecule quantitatively without an external electromagnetic field, e.g., UV light, which normally can damage tissue. Having been developed quite recently, this technique has evolved rapidly. Here, different bioluminescent proteins have been reviewed. In addition to a multitude of bioluminescent proteins, this manuscript focuses on the recent development of BRET sensors by utilizing quantum dots. The special size-dependent properties of quantum dots have made the BRET sensing technique attractive for the real-time monitoring of the changes of target molecules and bioimaging in vivo. This review offers a look into the basis of the technique, donor/acceptor pairs, experimental applications and prospects.

scholarly journals Design and Application of Highly Responsive Fluorescence Resonance Energy Transfer Biosensors for Detection of Sugar in Living Saccharomyces cerevisiae Cells

2007 ◽  
Vol 73 (22) ◽  
pp. 7408-7414 ◽  
Author(s):  
Jae-Seok Ha ◽  
Jae Jun Song ◽  
Young-Mi Lee ◽  
Su-Jin Kim ◽  
Jung-Hoon Sohn ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Signal Intensity ◽  
Binding Proteins ◽  
Dynamic Range ◽  
Binding Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy

ABSTRACT A protein sensor with a highly responsive fluorescence resonance energy transfer (FRET) signal for sensing sugars in living Saccharomyces cerevisiae cells was developed by combinatorial engineering of the domain linker and the binding protein moiety. Although FRET sensors based on microbial binding proteins have previously been created for visualizing various sugars in vivo, such sensors are limited due to a weak signal intensity and a narrow dynamic range. In the present study, the length and composition of the linker moiety of a FRET-based sensor consisting of CFP-linker1-maltose-binding protein-linker2-YFP were redesigned, which resulted in a 10-fold-higher signal intensity. Molecular modeling of the composite linker moieties, including the connecting peptide and terminal regions of the flanking proteins, suggested that an ordered helical structure was preferable for tighter coupling of the conformational change of the binding proteins to the FRET response. When the binding site residue Trp62 of the maltose-binding protein was diversified by saturation mutagenesis, the Leu mutant exhibited an increased binding constant (82 μM) accompanied by further improvement in the signal intensity. Finally, the maltose sensor with optimized linkers was redesigned to create a sugar sensor with a new specificity and a wide dynamic range. When the optimized maltose sensors were employed as in vivo sensors, highly responsive FRET images were generated from real-time analysis of maltose uptake of Saccharomyces cerevisiae (baker's yeast).

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