Date of Award

Spring 2013

Degree Type

Masters Thesis

Degree Name

Master of Science (MS)

Department

Chemistry and Biochemistry

School

Mathematics and Natural Sciences

Committee Chair

Sabine Heinhorst

Committee Chair Department

Chemistry and Biochemistry

Committee Member 2

Gordon Cannon

Committee Member 2 Department

Chemistry and Biochemistry

Abstract

Carboxysomes are specialized organelles that are filled with ribulose 1,5- bisphosphate carboxylase/oxygenase (RubisCO) needed for CO2 fixation. The major carboxysome shell proteins, CsoSl A, B, and C, form hexamers which tile together to form the facets of the thin shell. Each hexamer has a small central pore that may play a role in metabolite flux. Using predicted structural models, a specific amino acid within the conserved hexamer pore motif (Phe-Val-Gly-Gly-Gly-Tyr) was chosen to change the size and charge of the pore, respectively. Two mutants of Halothiobacillus neapolitanus were generated in which the wild type csoSJA gene was replaced with the desired mutant allele. The first mutant, G42L, contained a Leu residue at position 42 (Phe-Val-Leu-GlyGly- Tyr) and was predicted to have a greatly reduced pore. Mutant F40D (Asp-Val-GlyGly- Gly-Tyr) was predicted to have CsoS 1 A hexamer pores of opposite surface charge. The growth of the pore mutant cultures determined if the altered hexamer pores affected carboxysome function in preparation for the future assessment of diffusion of RubisCO metabolites across the shell. The H neapolitanus G42L pore mutant grew at a significantly slower rate than wild type in ambient air. The H neapolitanus F40D pore mutant had a different growth rate to wild type H neapolitanus cells in ambient air. Carboxysome were purified from both pore mutants and the polypeptide composition of the carboxysomes was compared using Western blots with antibodies specific for certain carboxysomal polypeptides. Both mutant organelles had an increased amount of CsoS ID when compared to wild type carboxysomes. Electron micro graphs of purified carboxysomes from each pore mutant and wild type were compared. The size and shape of mutant and wild type carboxysomes were compared by electron microscopy. Based on the results for the F40D and G42L mutant growth curves, changing the pore does appear to affect the function of the carboxysomes.

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