Molecular Basis for Iron Stress in Thermophilic Microorganisms

Iron is an essential element for life and iron homeostasis is major metabolic requirement.  Iron is found at the active centers of many redox enzymes, such as those of the electron transport chain, and therefore plays a central role as a cofactor for the metabolic processes supporting life.   However, iron is also extremely toxic when in the presence of oxygen, as it readily generates activated oxygen species (O₂–, H₂O₂, and •OH) that are deleterious to the cell We are undertaking both a chemical (structural and mechanistic) and a genetic approach to examining some of the key molecular species involved in the uptake, storage and regulation of Fe in hyperthermophilic microorganisms.  There are four target areas of this research

Dps proteins: We have identified, isolated and characterized a ferritin-like protein from Sulfolobus solfataricus, which assembles into a 12 subunit cage structure (Ss_Dps). Ferritins and bacterioferritins are 24 subunit assemblies that sequester Fe as a nanoparticle of rust, Fe₂O₃. Ss_Dps is a member of a family of proteins named Dps (for DNA binding protein from starved cells), which have been labeled as iron-storage proteins because of their similarity to ferritins and bacterioferritins. An exhaustive search of the Sulfolobus solfataricus genomic database suggests that there might not be an analog of the bacterial/mammalian ferritins in this archaeon.  Structural and phylogenetic analysis of the Ss_Dps suggests that it is different from other previously characterized Dps proteins with a unique di-Fe binding motif in the active site of the protein.  Ss_Dps is upregulated significantly in response to elevated H₂O₂ but not significantly with Fe.  This suggests a primary role for the Dps in oxidative stress rather than Fe storage/detoxification.

To probe the responses to H₂O₂ at an organismal level we have undertaken a study of the transcriptome using microarrays to probe the level of mRNA produced in response to oxidative and Fe stress (in collaboration with Drs.Mark Young and Martin Lawrence).  In addition, in collaboration with Dr.Brian Bothner we are probing the proteome of Sulfolobus using protein digestion and mass spectrometry to understand regulation in response to oxidative (and Fe) stress.

Fur:  The Ferric Uptake Regulatory (FUR) protein from bacterial systems has a highly conserved homolog in Sulfolobus solfataricus, which we have cloned into an E. coli heterologous expression system.  We are investigating the DNA binding of the purified protein.  In collaboration with Dr. Martin Lawrence we are crystallizing the protein for X-ray structural analysis.

Nanotechnology:  Some of the impetus for our work is the application of hyperthermophilic protein to the area of nanotechnology.  The protein cages derived from viruses and ferritin-like proteins are ideally suited as size constrained reaction environments for encapsulation of both inorganic and organic nanomaterials.  Increased thermal stability widens the synthetic window in which these proteins can be used in nanotechnology.  These protein-encapsulated nano-materials have application in biomedical imaging and drug delivery, magnetic recording, and catalysis.

Current Laboratory Personnel

Mark Allen, Ph.D. Student
Michelle Flenniken, Ph.D. Student
Seth Staples, Ph.D. Student
Zach Varpness, Ph.D. Student
Blake Wiedenheft, Ph.D. Student
Mackenzie Parker, Undergraduate Student

TBI scientists Trevor Douglas and Mark Young take a sample in Yellowstone

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