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).

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 In Vivo Self-Interaction of Nodavirus RNA Replicase Protein A Revealed by Fluorescence Resonance Energy Transfer

2005 ◽  
Vol 79 (14) ◽  
pp. 8909-8919 ◽  
Author(s):  
Billy T. Dye ◽  
David J. Miller ◽  
Paul Ahlquist
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Mitochondrial Membrane ◽  
Rna Viruses ◽  
Protein A ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Rna Replication ◽  
Outer Mitochondrial Membrane

ABSTRACT Flock house virus (FHV) is the best-characterized member of the Nodaviridae, a family of small, positive-strand RNA viruses. Unlike most RNA viruses, FHV encodes only a single polypeptide, protein A, that is required for RNA replication. Protein A contains a C-proximal RNA-dependent RNA polymerase domain and localizes via an N-terminal transmembrane domain to the outer mitochondrial membrane, where FHV RNA replication takes place in association with invaginations referred to as spherules. We demonstrate here that protein A self-interacts in vivo by using flow cytometric analysis of fluorescence resonance energy transfer (FRET), spectrofluorometric analysis of bioluminescence resonance energy transfer, and coimmunoprecipitation. Several nonoverlapping protein A sequences were able to independently direct protein-protein interaction, including an N-terminal region previously shown to be sufficient for localization to the outer mitochondrial membrane (D. J. Miller and P. Ahlquist, J. Virol. 76:9856-9867, 2000). Mutations in protein A that diminished FRET also diminished FHV RNA replication, a finding consistent with an important role for protein A self-interaction in FHV RNA synthesis. Thus, the results imply that FHV protein A functions as a multimer rather than as a monomer at one or more steps in RNA replication.

scholarly journals Spatial Distribution and Function of Sterol Regulatory Element-Binding Protein 1a and 2 Homo- and Heterodimers by In Vivo Two-Photon Imaging and Spectroscopy Fluorescence Resonance Energy Transfer

2005 ◽  
Vol 25 (8) ◽  
pp. 2946-2956 ◽  
Author(s):  
Aikaterini Zoumi ◽  
Shrimati Datta ◽  
Lih-Huei L. Liaw ◽  
Cristen J. Wu ◽  
Gopi Manthripragada ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Regulatory Element ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Two Photon ◽  
Sterol Regulatory Element ◽  
Photon Imaging ◽  
Imaging And Spectroscopy

ABSTRACT Sterol regulatory element-binding proteins (SREBPs) are a subfamily of basic helix-loop-helix-leucine zipper proteins that regulate lipid metabolism. We show novel evidence of the in vivo occurrence and subnuclear spatial localization of both exogenously expressed SREBP-1a and -2 homodimers and heterodimers obtained by two-photon imaging and spectroscopy fluorescence resonance energy transfer. SREBP-1a homodimers localize diffusely in the nucleus, whereas SREBP-2 homodimers and the SREBP-1a/SREBP-2 heterodimer localize predominantly to nuclear speckles or foci, with some cells showing a diffuse pattern. We also used tethered SREBP dimers to demonstrate that both homo- and heterodimeric SREBPs activate transcription in vivo. Ultrastructural analysis revealed that the punctate foci containing SREBP-2 are electron-dense nuclear bodies, similar or identical to structures containing the promyelocyte (PML) protein. Immunofluorescence studies suggest that a dynamic interplay exists between PML, as well as another component of the PML-containing nuclear body, SUMO-1, and SREBP-2 within these nuclear structures. These findings provide new insight into the overall process of transcriptional activation mediated by the SREBP family.

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