Time-Resolved Fluorescence Energy Transfer Measurements Between Site-Specific Probes On The Ca-Atpase Of Sarcoplasmic Reticulum In Different Enzymatic States

1988 ◽  
Author(s):  
Thomas C. Squier ◽  
Diana J. Bigelow ◽  
Jorge Garcia de Ancos ◽  
James E. Bishop ◽  
Giuseppe Inesi
Keyword(s):  
Energy Transfer ◽  
Sarcoplasmic Reticulum ◽  
Fluorescence Energy Transfer ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Site Specific ◽  
Fluorescence Energy

scholarly journals A High-Throughput Time-Resolved Fluorescence Energy Transfer Assay to Screen for Modulators of RGS7/Gβ5/R7BP Complex

2018 ◽  
Vol 16 (3) ◽  
pp. 150-161 ◽  
Author(s):  
Brian S. Muntean ◽  
Dipak N. Patil ◽  
Franck Madoux ◽  
James Fossetta ◽  
Louis Scampavia ◽  
...  
Keyword(s):  
Energy Transfer ◽  
High Throughput ◽  
Fluorescence Energy Transfer ◽  
Time Resolved Fluorescence ◽  
Throughput Time ◽  
Time Resolved ◽  
Fluorescence Energy

scholarly journals Establishment of Novel Protein Interaction Assays between Sin3 and REST Using Surface Plasmon Resonance and Time-Resolved Fluorescence Energy Transfer

2021 ◽  
Vol 22 (5) ◽  
pp. 2323
Author(s):  
Masamitsu Harada ◽  
Jun Nagai ◽  
Riho Kurata ◽  
Xiaofeng Cui ◽  
Takayuki Isagawa ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Protein Interaction ◽  
Plasmon Resonance ◽  
Fluorescence Energy Transfer ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Repressor Element ◽  
Fluorescence Energy

Repressor element-1 (RE-1) or neural restrictive silencer element (NRSE) bound with a zinc finger transcription repressor, RE-1 silencing transcription factor (REST, also known as neural restrictive silencer factor, NRSF) has been identified as a fundamental repressor element in many genes, including neuronal genes. Genes regulated by REST/NRSF regulate multifaceted neuronal phenotypes, and their defects in the machinery cause neuropathies, disorders of neuron activity), autism and so on. In REST repressions, the N-terminal repressor domain recruits Sin3B via its paired amphipathic helix 1 (PAH1) domain, which plays an important role as a scaffold for histone deacetylase 1 and 2. This machinery has a critical role in maintaining neuronal robustness. In this study, in order to establish protein–protein interaction assays mimicking a binding surface between Sin3B and REST, we selected important amino acids from structural information of the PAH1/REST complex and then tried to reconstitute it using recombinant short peptides derived from PAH1/REST. Initially, we validated whether biotinylated REST interacts with glutathione S-transferase (GST)-tagged PAH1 and whether another PAH1 peptide (PAH1-FLAG) competitively binds with biotinylated REST using surface plasmon resonance (SPR). We observed a direct interaction and competitive binding of two PAH1 peptides. Secondly, in order to establish a high-throughput and high-dynamic-range assay, we utilized an easily performed novel time-resolved fluorescence energy transfer (TR-FRET) assay, and closely monitored this interaction. Finally, we succeeded in establishing a novel high-quality TR-FRET assay and a novel interaction assay based on SPR.

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