Monitoring iron-sulfur cluster content of human glutaredoxin 2 using fluorescence proteins

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dc.contributor.advisorSilberg, Jonathanen_US
dc.creatorLi, Ruien_US
dc.date.accessioned2018-12-03T18:31:08Zen_US
dc.date.available2018-12-03T18:31:08Zen_US
dc.date.issued2008en_US
dc.description.abstractCurrent methods for directly determining whether an iron-sulfur cluster has been synthesized on a protein in vivo typically require that the protein of interest is purified prior to analysis for bound metallocluster. To establish an assay that can measure Fe/S-cluster synthesis in complex mixtures of biomolecules, we have fused a variety of fluorescent proteins (FP=BFP, CFP, GFP, and YFP) to human glutaredoxin 2 (Grx2) and characterized the effect of Grx2 metallocluster coordination on protein fluorescence. Gel filtration analysis revealed that FP-Grx2 fusion proteins are produced as a mixture of monomers and dimers when expressed in Escherichia coli , like native Grx2. The dimeric FP-Grx2 exhibited absorbance and circular dichroism spectra consistent with the presence of a Grx2-bound [2Fe2S] cluster, whereas the monomeric form of these fusion proteins lacked any detectable chromophores. Fluorescence analysis of the FP orthologs in these fusion proteins revealed that [2Fe2S]-cluster coordination produces a 30 to 50% reduction in FP fluorescence emission. These fluorescence indicators are expected to be useful for continuously monitoring Fe/S-cluster assembly on Grx2 in complex protein mixtures and should be useful for discovery of the proteins that mediate the assembly of metallocluster on Grx2 isoforms that are localized to both the nucleus and mitochondria.en_US
dc.format.extent56 ppen_US
dc.identifier.callnoTHESIS BIOCHEM. 2008 LIen_US
dc.identifier.citationLi, Rui. "Monitoring iron-sulfur cluster content of human glutaredoxin 2 using fluorescence proteins." (2008) Master’s Thesis, Rice University. <a href="https://hdl.handle.net/1911/103521">https://hdl.handle.net/1911/103521</a>.en_US
dc.identifier.digital304506979en_US
dc.identifier.urihttps://hdl.handle.net/1911/103521en_US
dc.language.isoengen_US
dc.rightsCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.en_US
dc.subjectMolecular biologyen_US
dc.subjectBiochemistryen_US
dc.subjectPure sciencesen_US
dc.subjectBiological sciencesen_US
dc.titleMonitoring iron-sulfur cluster content of human glutaredoxin 2 using fluorescence proteinsen_US
dc.typeThesisen_US
dc.type.materialTexten_US
thesis.degree.departmentBiochemistry and Cell Biologyen_US
thesis.degree.disciplineNatural Sciencesen_US
thesis.degree.grantorRice Universityen_US
thesis.degree.levelMastersen_US
thesis.degree.nameMaster of Artsen_US
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