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dc.contributor.advisorPerlstein, Deborahen_US
dc.contributor.advisorWhitty, Adrianen_US
dc.contributor.authorGrossman, John Daviden_US
dc.date.accessioned2021-02-04T19:03:06Z
dc.date.available2021-02-04T19:03:06Z
dc.date.issued2020
dc.identifier.urihttps://hdl.handle.net/2144/41987
dc.description.abstractIron sulfur (FeS) clusters are ubiquitous metallocofactors required by a large number of proteins involved in myriad cellular processes. Nuclear and cytosolic FeS proteins depend on the cytosolic iron sulfur cluster assembly (CIA) pathway for cluster acquisition. The CIA pathway begins with a scaffolding complex, comprising Nbp35 and Cfd1 in Saccharomyces cerevisiae. Nbp35 and Cfd1 each harbor a deviant Walker A domain for nucleotide hydrolysis that is essential for their FeS cluster scaffolding activity. Since there is little information about the CIA scaffold’s nucleotide hydrolysis activity, it has been challenging to discern the role nucleotide is playing in FeS cluster biogenesis. This thesis investigates the nucleotide driven steps of FeS cluster assembly and transfer, and the individual roles of the scaffold subunits Nbp35 and Cfd1. First addressed was answering the question of why two different scaffold subunits are needed for CIA function, and identifying the scaffold’s quaternary structure. Size exclusion chromatography revealed that the CIA scaffold exists as homodimers and heterodimers. Only Nbp352 and Nbp35-Cfd1 exhibited detectable ATPase activity. Though Cfd12 did not have detectable ATPase activity, it bound nucleotide with an affinity comparable to Nbp352 and Nbp35-Cfd1. Site directed mutagenesis and nucleotide binding studies revealed that the Cfd1 subunit is the high affinity binding site for ATP in Nbp35-Cfd1, and that the Nbp35 subunit binds nucleotide at saturating concentrations. Cfd1 therefore controls nucleotide binding in Nbp35-Cfd1. Additionally, it was found that the Cfd1 subunit is hydrolysis competent when complexed with Nbp35, identifying Nbp35 as an activator of Nbp35-Cfd1’s ATPase activity. Next, ATP’s role in FeS cluster biogenesis by CIA was identified. Mutation of the ATPase domain of Nbp35 impaired the ability of the scaffold to assemble and transfer FeS clusters in vivo. Four phenotypes were identified by observing how each mutation affected the scaffold’s nucleotide binding and hydrolysis. In vitro experiments established that cluster occupancy of the bridging cluster site of Nbp35-Cfd1 decreased the scaffold’s affinity for nucleotide. These results support a model of FeS cluster biogenesis in which nucleotide binding and FeS cluster binding regulate one other, with the bridging cluster site translating information to the ATPase site and vice versa. Nucleotide binding is also proposed to drive a conformational change that mediates interaction with another CIA component, later identified as Dre2. Dre2 was found to stimulate the rate of ATP hydrolysis by Nbp35-Cfd1 in an FeS cluster dependent manner. It is likely that nucleotide hydrolysis is then needed for the scaffold to assemble and/or transfer the FeS cluster. The results of these experiments have allowed us to describe the critical role of nucleotide in FeS biogenesis by CIA and explain the requirement for two distinct scaffold subunits. Finally, a fluorescent [Fe4S4] cluster sensor based on bacterial FNR (fumarate and nitrate reductase transcription factor) was designed, developed, and tested for practicality. FNR was fused to a SNAP tag protein which was then covalently labeled with a fluorescent molecule. The loss of cluster by the sensor resulted in an increase in fluorescence intensity, due to the cluster’s ability to quench fluorescence. As such, cluster decay rates could be measured as a function of increasing fluorescence intensity. The rates observed via fluorescence followed the same trends as the rates obtained by measuring the decay of clusters via absorbance. Encouragingly, the rates observed for the cluster decay were similar to decay rates determined previously via alternative methods.en_US
dc.language.isoen_US
dc.subjectBiochemistryen_US
dc.subjectATPen_US
dc.subjectCfd1en_US
dc.subjectCIAen_US
dc.subjectFeSen_US
dc.subjectNbp35en_US
dc.subjectScaffolden_US
dc.titleInvestigating the function of ATP hydrolysis during cluster biogenesis by the yeast cytosolic iron sulfur cluster assembly scaffolden_US
dc.typeThesis/Dissertationen_US
dc.date.updated2021-02-04T05:01:40Z
etd.degree.nameDoctor of Philosophyen_US
etd.degree.leveldoctoralen_US
etd.degree.disciplineChemistryen_US
etd.degree.grantorBoston Universityen_US
dc.identifier.orcid0000-0001-9586-0369


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