In Vivo Performance Evaluation of a Transdermal Near- Infrared Fluorescence Resonance Energy Transfer Affinity Sensor for Continuous Glucose Monitoring

2006 ◽  
Vol 8 (3) ◽  
pp. 296-311 ◽  
Author(s):  
Ralph Ballerstadt ◽  
Colton Evans ◽  
Ashok Gowda ◽  
Roger McNichols
Keyword(s):  
Performance Evaluation ◽  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Continuous Glucose Monitoring ◽  
Near Infrared ◽  
Resonance Energy Transfer ◽  
Glucose Monitoring ◽  
Resonance Energy ◽  
Near Infrared Fluorescence

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 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.

Sign in / Sign up

Export Citation Format

Share Document