Event Title

Measuring Heat Related to the Disassembly and Reassembly of Ferritin using Isothermal Titration Calorimetry

Poster Number

102

Session Title

Biology and Biomedical Research

Document Type

Poster Presentation

Faculty Mentor

Nicholas Grossoehme, Ph.D., and F. Wayne Outten, Ph.D., University of South Carolina

College

College of Arts and Sciences

Department

Department of Chemistry, Physics, and Geology

Description

Ferritin is an iron storage protein responsible for the accumulation of excess intracellular iron. Native ferritin self-aggregates into a nanocage structure containing a ferroxidase center that regulates the uptake and release of iron. In recent years, researchers have begun to explore using ferritin as a component in drug delivery. Ferritin is an attractive candidate because it is a native human protein that has the ability to encapsulate small molecules. Furthermore, it can be chemically or genetically modified to target very specific cells. One major limitation of drug delivery by ferritin lies in its inherent stability: harshly acidic conditions are needed to drive the disassembly of the nanocage. It was recently discovered that replacing the E-helix of human light chain ferritin with a GALA peptide repeat (hFtnL-GALA) would allow for the pH-induced disassembly to occur at a pH below 6, thus rendering ferritin a more attractive drug carrier under physiologically relevant conditions. This project aims to express and purify hFtnL-GALA with a subsequent thermodynamic characterization of the disassembly and reassembly of the nanocage. The chimeric protein is largely isolated in an insoluble form; consequently, the published protocol failed to produce enough protein for subsequent experiments. An alternate protocol was developed that leveraged urea to resuspend the insoluble fraction, followed by slow dilution to allow the protein to fold properly. Chromatographic analysis of the sample was consistent with an intact nanocage structure. Ongoing efforts are focused on developing a strategy that yields sufficient protein for further analysis.

Previously Presented/Performed?

Southeast Regional Meeting of the American Chemical Society (SERMACS), Savannah, Georgia, October 2019; SC INBRE Science Symposium, Columbia, South Carolina, January 2020; Sixth Annual Showcase of Undergraduate Research and Creative Endeavors (SOURCE), Winthrop University, April 2020

Grant Support?

Supported by a CRP grant from the South Carolina EPSCoR/IDeA Program, and by an SC INBRE grant from the National Institute for General Medical Sciences (NIH-NIGMS)

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Apr 24th, 12:00 AM

Measuring Heat Related to the Disassembly and Reassembly of Ferritin using Isothermal Titration Calorimetry

Ferritin is an iron storage protein responsible for the accumulation of excess intracellular iron. Native ferritin self-aggregates into a nanocage structure containing a ferroxidase center that regulates the uptake and release of iron. In recent years, researchers have begun to explore using ferritin as a component in drug delivery. Ferritin is an attractive candidate because it is a native human protein that has the ability to encapsulate small molecules. Furthermore, it can be chemically or genetically modified to target very specific cells. One major limitation of drug delivery by ferritin lies in its inherent stability: harshly acidic conditions are needed to drive the disassembly of the nanocage. It was recently discovered that replacing the E-helix of human light chain ferritin with a GALA peptide repeat (hFtnL-GALA) would allow for the pH-induced disassembly to occur at a pH below 6, thus rendering ferritin a more attractive drug carrier under physiologically relevant conditions. This project aims to express and purify hFtnL-GALA with a subsequent thermodynamic characterization of the disassembly and reassembly of the nanocage. The chimeric protein is largely isolated in an insoluble form; consequently, the published protocol failed to produce enough protein for subsequent experiments. An alternate protocol was developed that leveraged urea to resuspend the insoluble fraction, followed by slow dilution to allow the protein to fold properly. Chromatographic analysis of the sample was consistent with an intact nanocage structure. Ongoing efforts are focused on developing a strategy that yields sufficient protein for further analysis.