1. Profile of two carboxyosome subunits of Synechocystis Todd O. Yeates Nam Tonthat

2. Goal To profile two carboxysome hexamers of Synechocystis, which were recently structurally solved by the Yeates Lab.

3. Carboxysomes Inclusion bodies Present in cyanobacteria and many chemoautotrophs Filled with RuBisCO RuBisCO fixes inorganic carbons (HCO 3 -, CO 2 ) to an organic form (PGA) RuBisCO lacks specificity for CO 2 and O 2 RuBisCO is less efficient, compared to other typical enzymes Working hypothesis:  Carbon fixation can be optimized by localizing RuBisCO in a CO 2 rich environment *Badger, Murray. Price, Dean. 2003

4. Carboxysome shells Shell subunits are small, homologous proteins which form hexamers or pentamers  Hexamers/Pentamers form sheets Two such subunits in Synechocystis:  ccmk2 and ccmk4 A: ccmk2 B: ccmk4 *Kerfeld, Sawaya, Tanka, Phillips, Beeby, Yeates 2005

5. Red=>ccmk2 Blue=>ccmk4 ccmk2 & ccmk4 Overall folding of ccmk2 & ccmk4 is practically identical Major structural difference lies in the orientation of the fourth helix  Pro97 (ccmk4)  Glu97 (ccmk2) *Kerfeld, Sawaya, Tanka, Phillips, Beeby, Yeates 2005

6. Metabolites Important metabolites: O 2, CO 2, HCO 3 - RuBisCO is unable to distinguish between CO 2 and O 2 In Synechocystis and several other cyanobacteria:  several mechanisms of transporting HCO 3 - and CO 2 have been uncovered*  HCO 3 - is the form accumulated in the cytoplasm*  Mass spectrometry work has shown Carbonic Anhydrase activity in carboxysome ** Carbonic Anhydrase is the enzyme used to convert HCO 3 - to CO 2 * Price and Badger 1989 **So and Espie1998

7. Hypothesis The pore created by the ccmk2/ccmk4 hexamer serves as a gateway for molecules to move through.

8. Steps for radius calculation 1. Starting with a position inside the pore at a given end 2. Find the largest sphere that will fit within the space bounded by surrounding atoms 3. Exhaustively explore all surrounding positions in plane 4. Move through the pore iteratively toward the other side

9. Pore radius: ccmk2 3.39 Å2.69 Å Z Y

10. Pore radius: ccmk4 Z Y 2.29 Å 1.77 Å

11. Electrostatics calculation “Ezprot”  program written by Frank Petit  wrapper for UHBD (University of Houston Brownian Dynamics) Used the Poisson Boltzmann equation to calculate the potential Describe electrostatic potential due to:  Non-homogeneous dielectric  Mobile counterions  “Fixed” (biomolecular) charge distribution

12. Closer look at the pore: ccmk2 Residues of the ccmk2 pore:  LYS36 ILE37 GLY38 SER39 *Kerfeld, Sawaya, Tanka, Phillips, Beeby, Yeates 2005

13. Charge distribution of the pore: ccmk2

14. Closer look at the pore: ccmk4 Residues of the ccmk4 pore:  ARG38 ALA39 GLY40 SER41 *Kerfeld, Sawaya, Tanka, Phillips, Beeby, Yeates 2005

15. Charge distribution of the pore: ccmk4

16. Concluding remarks It would be reasonable to speculate that the pore is indeed a semi-selective gateway for molecules to diffuse in and out based on  What we know about the components that are inside and around the carboxysome  Information about the size and charge of the pore

17. Future work There is still much to be done to fully elucidate the bacterial microcompartment’s role in the carbon dioxide concentrating mechanism  Internal organization of the carboxysome  Sequence of events that lead to the formation of the carboxysome Help to understand other microcomparments:*  Salmonella typhimurium, Klebsiella oxytoca, Escherichia coli  Presence of inclusion bodies when grown under anaerobic conditions with either ethanolamine or propanediol as the energy source *Stojiljkovic I, Baumler AJ, Heffron F. 1995

18. Thank-you SoCalBSI Yeates Lab:  Dr. Yeates  Yingssu Tsai