Title

Identification and Characterization of Protein Interactions in the Carboxysome of Halothiobacillus neapolitanus

Date of Award

2006

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry and Biochemistry

First Advisor

Gordon Cannon

Advisor Department

Chemistry and Biochemistry

Abstract

The identification and characterization of Halothiobacillus neapolitanus carboxysome protein interactions was continued for the purpose of understanding the role shell proteins may play in CO2 metabolism. Previous investigations of the carboxysome identified a possible interaction between the catalytic RuBisCO enzyme and the 3-4 nm protein shell (50, 96). In this study, carboxysome protein interactions were investigated in vitro by freeze-break, EDTA treatment and disulfide bond reduction to reveal two distinct types of carboxysome shell protein groups. Protein interactions observed in vitro were confirmed by yeast two-hybrid screens of carboxysome protein pairs. Analysis of interacting proteins resulted in a basic map that was used to propose a representative cross-section model of the carboxysome shell. Finally, carboxysome shell proteins were heterologously expressed in E. coli , purified and used to form carboxysome-like protein structures in vitro . The carboxysome-like protein particles had a 3-4 nm protein shell that was filled with RuBisCO molecules, which is characteristic of wild type carboxysomes. Furthermore, magnesium and carboxysomal RuBisCO were required components to form carboxysome-like protein structures in vitro . Analysis of carboxysome shell protein stoichiometric ratios and hypothetical carboxysome shell protein arrangements were discussed.