In vivo fluorescent detection of Fe-S clusters coordinated by human GRX2
| Autor: | Kevin G. Hoff, Stephanie J. Culler, Ryan M. McGuire, Jonathan J. Silberg, Christina D. Smolke, Peter Q. Nguyen |
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| Rok vydání: | 2009 |
| Předmět: |
PROTEINS
Iron Recombinant Fusion Proteins Clinical Biochemistry Mutant Biochemistry Cell Line 03 medical and health sciences Bimolecular fluorescence complementation 0302 clinical medicine Glutaredoxin Drug Discovery Humans Binding site Molecular Biology Glutaredoxins 030304 developmental biology Pharmacology 0303 health sciences Binding Sites biology Circular Dichroism Glutaredoxin 2 General Medicine Fluorescence Cell biology Cytosol Luminescent Proteins CHEMBIO Microscopy Fluorescence SIGNALING Mutation biology.protein Molecular Medicine RNA Interference ISCU Dimerization 030217 neurology & neurosurgery Sulfur |
| Zdroj: | Chemistrybiology. 16(12) |
| ISSN: | 1879-1301 |
| Popis: | SummaryA major challenge to studying Fe-S cluster biosynthesis in higher eukaryotes is the lack of simple tools for imaging metallocluster binding to proteins. We describe the first fluorescent approach for in vivo detection of 2Fe2S clusters that is based upon the complementation of Venus fluorescent protein fragments via human glutaredoxin 2 (GRX2) coordination of a 2Fe2S cluster. We show that Escherichia coli and mammalian cells expressing Venus fragments fused to GRX2 exhibit greater fluorescence than cells expressing fragments fused to a C37A mutant that cannot coordinate a metallocluster. In addition, we find that maximal fluorescence in the cytosol of mammalian cells requires the iron-sulfur cluster assembly proteins ISCU and NFS1. These findings provide evidence that glutaredoxins can dimerize within mammalian cells through coordination of a 2Fe2S cluster as observed with purified recombinant proteins. |
| Databáze: | OpenAIRE |
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