Bates, Maura

Johnson, Robin

Moore, Faculty Advisor, Laura

Digital Commons @ Illinois Wesleyan University

Illinois Wesleyan UniversityDigital Commons @ IWUJohn Wesley Powell Student ResearchConference 2006, 17th Annual JWP ConferenceApr 8th, 9:00 AM - 10:00 AMOxygen Sensing in the FNR Transcription Factor:Creation and Characterization of Variants withAerobically Stable [4FE-4S] ClustersRobin JohnsonIllinois Wesleyan UniversityMaura BatesIllinois Wesleyan UniversityLaura Moore, Faculty AdvisorIllinois Wesleyan UniversityFollow this and additional works at: http://digitalcommons.iwu.edu/jwprcThis Event is brought to you for free and open access by The Ames Library, the Andrew W. Mellon Center for Curricular and FacultyDevelopment, the Office of the Provost and the Office of the President. It has been accepted for inclusion in Digital Commons @ IWU bythe faculty at Illinois Wesleyan University. For more information, please contact digitalcommons@iwu.edu.©Copyright is owned by the author of this document.Robin Johnson; Maura Bates; and Laura Moore, Faculty Advisor, "Oxygen Sensing in the FNR Transcription Factor:Creation and Characterization of Variants with Aerobically Stable [4FE-4S] Clusters" (April 8, 2006). John Wesley PowellStudent Research Conference. Paper 40.http://digitalcommons.iwu.edu/jwprc/2006/posters/40THE JOHN WESLEY POWELL STUDENT RESEARCH CONfERENCE - APRIL 2006 Poster Presentation P7 OXYGEN SENSING IN THE FNR TRANSCRIPTION FACTOR: CREATION AND CHARACTERIZATION OF VARIANTS WITH AEROBICALLY STABLE [4FE-4S] CLUSTERS Robin Johnson. and Maura Bates and Laura Moore* Chemistry Department, Illinois Wesleyan University Activity of the FNR protein, a transcription factor in the facultative anaerobe Escherichia coli, is contingent upon the integrity of a [4Fe-4S] cluster incorporated by the protein's effector domain. The cluster is ligated by cysteine residues at positions 20, 23, 29, and 1 22. In wild-type FNR, this [4Fe-4SJ cluster degrades upon exposure to molecular oxygen, and the resulting conformational changes cause the homodimer to separate into monomers, deactivating the protein. A previous study has identified an FNR variant, Leu28His, which has an oxygen-stable [4Fe-4S] cluster. This mutant protein retains activity under aerobic conditions. Using beta-galactosidase assays to quantify in vivo FNR activity, our lab has explored the oxygen sensitivity of several other FNR variants with amino acid substitutions adjacent to ligating cysteines. While many of these proteins exhibit oxygen sensitivity similar to that of wild-type FNR, the mutants Leu28Lys, Leu28Asn, Leu28Gln, Leu28Ser, and Leu28Tyr all retain some activity under aerobic conditions. Western blot analysis suggests that this increase in activity as compared to wild-type is not due to an increase in protein concentration. These mutant proteins have been isolated and characterized by absorption spectroscopy. The spectra indicate that the [4Fe-4S] clusters of these FNR variants do exhibit some degree of oxygen stability. As only substitutions of amino acids with amine, alcohol, or amide side chains have so far resulted in aerobically active FNR, oxygen stability of the [Fe-4S] cluster may result from donation of a hydrogen bond at position 28. 

Oxygen Sensing in the FNR Transcription Factor: Creation and Characterization of Variants with Aerobically Stable [4FE-4S] Clusters

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Oxygen Sensing in the FNR Transcription Factor: Creation and Characterization of Variants with Aerobically Stable [4FE-4S] Clusters

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