Author

Mukkamala

Date of Award

12-2006

Degree Name

Master of Science

Department

Chemistry

First Advisor

Dr. Subra Muralidharan

Second Advisor

Dr. Brian C. Tripp

Third Advisor

Dr. Sherine Obare

Fourth Advisor

Dr. Dongil Lee

Access Setting

Masters Thesis-Open Access

Abstract

The structural protein flagellin, FlaA derived from hyperthermophilic Aquifex pyrophilus, a rod shaped eubacterial species that grows near hydrothermal vents at an optimum growth temperature of 85 °C forms elongated thermostable flagella nanotubes. FlaA was successfully over-expressed for the first time using pET28c plasmid in E. coli BL21 cells at 37 °C. The protein was isolated from inclusion bodies that were solubilized by alkaline pH, refolded by dialysis and purified by chromatographic techniques. The FlaA was characterized by fluorescence, light scattering and circular dichroism. The FlaA was not in the correct folded form to form flagella as efforts to form them employing temperature and salt resulted in random aggregates. Large rod shaped aggregates were obtained employing polyethylene glycol and were characterized by dynamic light scattering, fluorescence microscopy after staining with NanoOrange and transmission electron microscopy. The emission from the tryptophan residues were monitored in terms of intensity and anisotropy to understand the unfolding of the FlaA in the presence of guanidine hydrochloride (GdnHCl). The folded to completely unfolded state had an intermediate partially folded state with the completely unfolded state requiring 7 M GdnHCI. This work provides the basis for formation of thermally stable protein nanotubes for the generation of novel bionanostructures.

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