Exploring the Biology of Disulfide-Rich Hyperthermophiles through Protein Phylogenetic Profiles Navapoln Ramakul 1, Morgan Beeby 12, and Todd O. Yeates 123 Southern California Bioinformatics Summer Institute 1 Department of Chemistry and Biochemistry, 2 Department of Energy Center for Genomics and Proteomics, and 3 Molecular Biology Institute, University of California, Los Angeles, CA

UCLA Bioinformatics: Yeates Lab Goals: determine and analyze the three- dimensional structures of proteins. Research: focus on protein structure & function, protein sequence & evolution, and protein assembly & design. Methods: crystal structure determination through theoretical and computational methods.

General Overview Genomic Databases: create opportunities for new kinds of computational analyses and novel discoveries. Advantage: special comparative studies using multiple genomes to compare sequence vs. structure. Present Research: investigate the surprising revelation about disulfide bonds in certain microbes from comparative studies.

Protein Disulfide Bonds Previously believed to be prominent only outside the cell. Inside the cell Disulfides only rarely found. Disulfides are transient or functionally important, rather than stabilizing. Outside the cell Abundant. Intro Comput. Methods Results & Significance Applications & Future Directions Summary

Recent Studies Unexpected disulfides in an intracellular protein. Crystal structure of adenylosuccinate lyase (ASL) from the P. aerophilum surprisingly shown a protein chain stabilized by three disulfide bonds (Toth et al., JMB (2000) 301, ). Toth et al., JMB (2000) 301, Disulfide bond Intro Comput. Methods Results & Significance Applications & Future Directions Summary

Evidence for Abundant S-S bonds in P. aerophilum Mallick, Boutz, Eisenberg, and Yeates (2002). PNAS 99, Proteins with Even # of Cysteines Intro Comput. Methods Results & Significance Applications & Future Directions Summary

Disulfide Abundance in Various Genomes Genome f (S- S) Pyrobaculum aerophilum 0.44 Aeropyrum pernix 0.40 Pyrococcus abyssi 0.31 Pyrococcus horikoshii 0.28 Aquifex aeolicus 0.17 Methanobacterium thermo 0.15 Thermotoga maritima 0.13 Methanococcus jannasc 0.13 Archaeoglobus fulgidus 0.11 Mycoplasma genitalium 0.06 Synechocystis PCC Ureaplasma urealyticum 0.07 Neisseria meningitidis 0.06 Mycobacterium tubercu 0.07 Rickettsia prowazekii 0.06 Haemophilus influenzae 0.05 Escherichia coli 0.05 Treponema pallidum 0.03 Helicobacter pylori 0.03 Bacillus subtilis 0.01 Genome f (S-S) Pyro. aerophilum °C Aero. pernix °C Pyro. abyssi °C Pyro. horikoshii °C Aqui. aeolicus °C Meth. thermo °C Blue = archaea = thermophile 90°C 86°C 92°C Mallick, Boutz, Eisenberg, and Yeates (2002). PNAS 99, Archaeal branch Eubacterial branch

Exploring disulfide-rich hyperthermophiles Find the sequences of glutaredoxin-like protein in different organisms. Investigate the glutaredoxin-like protein in those disulfide-rich hyperthermophiles. Goals: differences between glutaredoxin-like protein in hyperthermophiles and glutaredoxin in organisms. Intro Comput. Methods Results & Significance Applications & Future Directions Summary

Why glutaredoxin-like protein? Only present among hyperthermophiles. Operates in thiol-disulfide reaction via CXXC motif which either form a disulfide (oxidized form) or a dithiol (reduced form). Requires for many functions including electron and proton transport to essential enzymes like ribonucleotide reductase. Involves in formation of disulfide bonds in protein folding. 90 o Prototypical fold: E.coli thioredoxin (2TRX.pdb)

Methods The sequences used in this study were obtained from the National Center for Biotechnology Information ( Obtain the control sequence of glutaredoxin (E. coli) to find glutaredoxin-like protein. Search for the glutaredoxin-like protein sequences of hyperthermophilic archaea. Using Sequence-Structure Mapping to identify potential disulfide bonds. Compare and analyze using multiple sequences alignment program, such as ClustalW, T-Coffee, or MSA. Intro Comput. Methods Results & Significance Applications & Future Directions Summary

Results ClustalW multiple sequences alignment of these glutaredoxin-like proteins shows two CXXC motifs Glutaredoxin-like protein has more than 85 amino acids. Green = P. aerophilum Black = Hyperthermophilic archeae Blue = Bacteria = CXXC motif Two CXXC motifs

Results Most organisms have 1 CXXC motif in glutaredoxin. Glutaredoxin-like protein has two redox-active CXXC motifs per polypeptide. Exception: P. aerophilum has only 1 CXXC motif. Intro Comput. Methods Results & Significance Applications & Future Directions Summary P. furiosus: 1A8L.pdb, Nat. Struct. Biol. (1998), 5 (7) J08.pdb, unpublished P. horikoshii: CXXC motifs

Limitation Only 25 genomes. Some glutaredoxin-like proteins have not yet been sequenced. Intro Comput. Methods Results & Significance Applications & Future Directions Summary

Applications and Further Studies: How disulfide bonds involve in protein folding? To identify disulfide-bonded protein-protein interactions and networks. To investigate the stability mechanisms by disulfide bonds. Intro Comput. Methods Results & Significance Applications & Future Directions Summary

Most of the hyperthermophiles have 2 CXXC motifs in order to have abundant disulfide bonds. The abundance of disulfide bonds appear to play a key role in stabilizing protein at high temperature. Intracellular disulfide bond is a characteristic of all archaea or an adaptation to high temperature. This study illustrates the power of integrating genomic data with protein structure and function to illuminate the chemistry and biology of unusual organisms. Intro Comput. Methods Results & Significance Applications & Future Directions Summary

Acknowledgements Yeates Lab Dr. Todd Yeates Morgan Beeby Everyone else at Yeates lab CalState LA Research mentors SoCalBSI Program SoCalBSI interns Support National Science Foundation (NSF) National Institutes of Health (NIH). UCLA-DOE Center for Genomics and Proteomics