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Showing 1 to 20 of 21 for “"FRET assay"”.
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Development And Optimization Of The First High Throughput In Vitro Fret Assay To Characterize The Saccharomyces Cerevisiae Gpi-T
<p>DEVELOPMENT AND OPTIMIZATION OF AN IN VITRO FRET ASSAY TO CHARACTERIZE THE SACCHAROMYCES CEREVISIAE GPI TRANSAMIDASE</p> <p>By</p> <p>SANDAMALI AMARASINGHA EKANAYAKA</p> <p>December 2013</p> <p>Advisor: Dr. Tamara L. Hendrickson</p> <p>Major: Biochemistry</p> <p>Degree: Doctor of Philosophy</p> …
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Determining protein interaction specificity of native and designed bZIP family transcription factors
… this technique, I developed a solution-based FRET assay. In this experiment, two different dye-labeled versions of each protein are purified and mixed together at multiple concentrations to generate binding curves that quantify the affinity of each pair-wise interaction. Using the array assay, …
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Development of Novel Chemical Biology Tools for Probing Structure-Function Relationships in G Protein-Coupled Receptors
… Then a fluorescence resonance energy transfer (FRET) assay was developed for the labeled Rho to demonstrate its functionality with respect to ligand binding. Rho consists of a chromophore ligand, 11-cis-retinal, bound to the opsin via a Schiff base bond. The photoisomerization of 11-cis-retinal …
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STRUCTURAL INSIGHTS INTO POLYUBIQUITIN CHAIN FORMATION MEDIATED BY NEDD4 HECT LIGASE
… Leveraging in-silico modeling and a novel TR-FRET-based ubiquitination assay, we pinpoint conserved HECT residues crucial for catalysis. Additionally, we dissected multiple determinants in the N-terminal domains of NEDD4 essential for intramolecular autoinhibition. Moreover, the establishment …
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Structural and mechanistic studies on antimicrobial peptides that target multi-drug resistant bacteria
… fluorescence resonance energy transfer (FRET) assay developed by the Caffrey group.
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Engineering Fluorescently Labeled Human Fibroblast Growth Factor One Mutants and Characterizing Their Photophysics Properties Towards Designing FRET Assays
… which can lead to a myriad of cancers. smFRET is an ultrasensitive distant dependent (1-10 nm) technique capable of resolving such heterogeneity in structural dynamics and binding affinities (Kd). Therefore, we successfully designed and characterized fluorescently labeled hFGF1 tracers …
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A FRET Investigation into Molecular Mechanisms of Cardiac Troponin Activation in Reconstituted Thin Filaments
… A multi-site Förster resonance energy transfer (FRET) assay in cardiac Tn in reconstituted thin filaments was used to investigate the Ca2+-dependent structure and dynamics of the SABS, and show current theories behind Tn activation are biased using structures developed in isolated Tn. The SABS …
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The Ligand and Membrane-binding Behaviour of the Phosphatidylinositol Transfer Proteins (PITPa & PITPb)
… using a fluorescence resonance energy transfer (FRET)-based assay. DPI investigations suggest that PITPβ had a two-fold higher affinity for membranes compared to PITPα. This was reflected by a four-fold faster ligand transfer rate for PITPβ in comparison to PITPα as determined by the FRET assay. …
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Investigation of the mechanism of transfer of a-tocopherol by the human a-tocopherol transfer protein (H-a-TTP) /
… of a fluorescence resonance energy transfer (FRET) assay. (/?)-2,5,7,8-tetramethyl-2-[9-(7-nitro-benzo[l,2,5]oxdiazol-4-ylamino)-nonyl]- chroman-6-ol (NBD-toc) has allowed for the development of the FRET-based ligand transfer assay. This ligand has been utilized in a series of experiments …
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Development of Methods for Structural Characterization of Pantoea stewartii Quorum-Sensing Regulator EsaR
… and Förster resonance energy transfer (FRET), experimental methods were designed for determining the dimerization interface of EsaR and for testing the hypothesis that EsaR undergoes a conformational shift when presented with the AHL ligand. To identify residues of the dimerization …
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Deciphering Underlying Mechanisms Of Photoreceptor Outer Segment Degradation; Involvement Of Intracellular Trafficking
… clearance of POS. Lack of molecular methods and assays have limited our knowledge of these factors. In this project I developed a novel method which is used to uncover new factors specifically involved in POS trafficking in the RPE. Foster Resonance Energy Transfer or FRET is an energy transfer …
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Single molecule fret study on the mechanism of RecA mediated strand exchange
… The study of strand exchange in ensemble assays is limited by the diffusion limited homology search process which masks the subsequent strand exchange reaction. We developed a single molecule fluorescence assay with a few basepair and milliseconds resolution which can separate initial …
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Using biophysical analysis to study the regulation of the budding yeast kinetochore
… this hypothesis, I developed a single molecule FRET assay to examine conformational changes of the complex while simultaneously monitoring its microtubule-binding activity. I uncovered a novel mechanism of regulation of the Ndc80 complex that is intrinsic to its structure; tight bending of the …
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Mechanism of tocopherol transfer by human α-tocopherol transfer protein (α-hTTP)
… a fluorescence resonance energy transfer (FRET) assay was developed to monitor the kinetics of ligand transfer by a-hTTP in lipid vesicles. Preliminary results implied that NBD-a-Toe simply diffused from 6-His-a-hTTP to acceptor membranes since the kinetics of transfer were not responsive …
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Single-molecule studies of protein degradation and kinesin-8 motility
… fluorescence resonance energy transfer (sm- FRET) assay revealed dynamic switching of ClpX subunit conformations between multiple states. In the absence of nucleotide, ClpX explored available conformational spaces thermally in an erratic manner. Nucleotide binding to ClpX hexamer leads to …
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Untersuchungen zur stimulierten GTPase von EF-Tu aus Thermus thermophilus
… modifiziert und gereinigt. Bei den, auf FRET-basierenden, Fluoreszenztitrationsexperimenten konnten die Affinitäten der fluoreszierenden EF-Tu Varianten zur Phe-tRNAPhe-X47F wegen einem starken Fluoreszenzhintergrund und dementsprechend schwachen Signal nur ungenau bestimmt werden. Das …
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Modular design of coiled coils to target bZIP transcription factors
… peptide and target bZIP. Using a solution FRET assay to test both the original and redesigned peptide's affinity for the target and 30 off-target bZIPs, I showed that redesigning the solvent-exposed positions did stabilize the design-target interaction from 3-fold to 90-fold but the …
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Mechanical manipulation and visualization of single biomolecules using fluorescence spectroscopy
… To address this, we employed an integrated force-FRET (Fluorescence resonance energy transfer) assay to interrogate single biomolecules under applied forces between ~ 0.3 and 28 pN. In human cells, the ends of chromosomes are capped by 100-200 nt long telomeric DNA, consisting of tandem …
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Developing high-resolution methods to study DNA and RNA dynamics
… several fluorescence-based high-resolution assays to study DNA and RNA dynamics in vitro and in vivo. First, we established a single molecule FRET assay to study homodimeric single-stranded binding protein (Thermus thermophilus SSB) specifically binding and protecting single-stranded DNA …
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Design and synthesis of novel non peptidomimtic beta-secretase inhibitors in the treatment of Alzheimer's disease
… evaluated for BACE1 inhibitory activity in a FRET (fluorescence resonance energy transfer) enzymatic assay (test A). With the aim to enhancing the biological activity of the lead compound, we applied the molecular simplification approach to design and synthesize novel heterocyclic compounds …
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