1. Computational studies of intramolecular disulfide bonded catenanes as a novel stabilizing mechanism in thermophilic microbes August 23, 2007 Daniel Park Yeates lab, MBI, UCLA SoCalBSI

2. Today Intracellular disulfide abundance in thermophiles/hyperthermophiles P. aerophilum citrate synthase Searching for catenanes Results

3. Importance of studying thermophilic enzymes Industrial applications –Engineering heat-stable biomolecules –Utilizing those found in nature Taq DNA polymerase for PCR Insight into protein folding mechanisms –Evolution of thermostable proteins

4. Intracellular disulfide bond abundance Mallick et al., 2002 PNAS 99, pp. 9679-9684

5. Presence of disulfide bonds within the intracellular proteins of P. aerophilum Both lanes reduced Presense and absence of iodoacetamide Large fraction of P. aerophilum proteins contain disulfide bonds Boutz et al., 2007 JMB 368, pp. 1332-1344

6. Citrate synthase (PaCS) from P. aerophilum Boutz et al., 2007 JMB 368, pp. 1332-1344

7. Catenane structure of PaCS Boutz et al., 2007 JMB 368, pp. 1332-1344

8. Disulfide bonds: contribution to the thermostability of PaCS Boutz et al., 2007 JMB 368, pp. 1332-1344

10. Cysteine abundance at terminal regions

11. Alignment of thermophilic citrate synthase

12. Approach

13. Possible catenanes by temperature

14. Cysteine abundance at terminal regions

15. Clusters of orthologous groups (COG) functional classifications INFORMATION STORAGE AND PROCESSING [J] Translation, ribosomal structure and biogenesis [A] RNA processing and modification [K] Transcription [L] Replication, recombination and repair [B] Chromatin structure and dynamics CELLULAR PROCESSES AND SIGNALING [D] Cell cycle control, cell division, chromosome partitioning [Y] Nuclear structure [V] Defense mechanisms [T] Signal transduction mechanisms [M] Cell wall/membrane/envelope biogenesis [N] Cell motility [Z] Cytoskeleton [W] Extracellular structures [U] Intracellular trafficking, secretion, and vesicular transport [O] Posttranslational modification, protein turnover, chaperones METABOLISM [C] Energy production and conversion [G] Carbohydrate transport and metabolism [E] Amino acid transport and metabolism [F] Nucleotide transport and metabolism [H] Coenzyme transport and metabolism [I] Lipid transport and metabolism [P] Inorganic ion transport and metabolism [Q] Secondary metabolites biosynthesis, transport and catabolism POORLY CHARACTERIZED [R] General function prediction only [S] Function unknown

16. Possible microbial catenanes by function

18. Possible thermophilic catenanes by function

19. Possible thermophilic catenanes further classified by COGs (top 7) Functional classification COG typeNo. of catenanes [O] Posttranslational modification, protein turnover, chaperones Peroxiredoxin7 [C] Energy production and conversion Citrate synthase5 [C] Energy production and conversion Anaerobic dehydrogenase, typically selenocysteine-containing 5 [?] Unclassified5 [G] Carbohydrate transport Transketolase, N-terminal subunit4 [EP] Amino acid and inorganic ion transport ABC-type dipeptide/oligopeptide/nickel transport system 4 [E] Amino acid transport 3-dehydroquinate synthetase4

20. Possible catenane among peroxiredoxin homologs? [O] COG0450 Peroxiredoxin (7) Thermoanaerobacter tengcongensis MB420808569 Methanosaeta thermophila PT116754713 Pyrobaculum islandicum DSM 4184119873344 Pyrobaculum islandicum DSM 4184119871684 Pyrobaculum calidifontis JCM 11548126458809 Pyrobaculum arsenaticum DSM 13514145590729 Methanocaldococcus jannaschii DSM 266115668917 [C] COG0372 Citrate synthase (5) Pyrobaculum islandicum DSM 4184119873179 Pyrobaculum calidifontis JCM 11548126459178 Pyrobaculum arsenaticum DSM 13514145592430 Pyrobaculum aerophilum str. IM218312809 Aeropyrum pernix K114601576

21. P. islandicum DSM 4184 peroxidase: alignment with homologs

22. P. islandicum peroxidase homolog

24. Future directions MD simulations of possible catenanes Determine structures of most likely catenanes by X-ray crystallography Investigate correlation between psychrophilic proteins and disulfide bonding

25. Acknowledgements Todd Yeates Neil King Jason Forse Brian O’Connor Jamil Momand Sandra Sharp Wendie Johnston Nancy Warter-Perez SoCalBSI program Ronnie Cheng Funded by NIH, NSF, EWD, DOE