Bioinformatics Institute 2012 Group Project Sara Volk (OLLU, Biology)

1. Thiomicrospiracrunogena:Where biology, chemistry, and physics collaborateA story of ABC transporters and biochemical pathways Bioinformatics Institute 2012 Group Project Sara Volk (OLLU, Biology) TeresitaMunguia (OLLU, Chemistry) Troy Messina (Centenary College, Biophysics)

2. Background • Thiomicrospiracrunogena (T.cru) • Sulfur-oxidizing chemoautolithotroph • ABC transporters are an estimated 75% of transporter genes in genome) http://genome.jgi-psf.org/thicr/thicr.home.html http://blog.mywonderfulworld.org/assets/hydrothermal-vent

3. Some of the ABC transporters in T.cru (copied from KEGG) Each transporter is encoded by 3-5 genes (TM subunits and the ATPases are dimers that can be homodimers or heterodimers)

4. Courses Impacted • Introductory Biology Courses (Sara) • Summer Undergraduate Research (Terry) • Biochemistry Course (Terry) • Upper-level Biophysics course (Troy)

5. Our Project Plans • Create data set of ABC transporter genes • Mine data set for intro biology class (Sara) • Question: Predict which subunit based on similarity and signal seq/TM prediction • Look at biochemical pathways; see if can easily tie to ABC tx (Terry) • Question: ? • Examine conservation of periplasmic binding proteins in T.cru(Troy) • Conserved components will be important for folding and function

6. Upper Level Biophysics Course: Learning Objectives (Troy) • Structure/function relationships • Homology and conservation due to similar function • Applications of thermodynamics, i.e. Nernst Equation • Bioinformatics Module • Does structural similarity give behavioral similarity? • Can we build models for fold and function families?

7. What is an ABC transporter? • Use KEGG (http://www.genome.jp/kegg/) to look at the huge list of ABC transporters • Many have three components. Why?

8. What do the genes encode? • Subunits of ABC transporter • Periplasmic solute-binding protein • Transmembrane ‘channel’ protein • ATP-hydrolyzing cytoplasmic component http://www.biologie.uni-regensburg.de/Mikrobio/Thomm/Buttons/reg_fig1.gif

9. Transmembrane prediction: Hydrophobicity ‘motif’ • Is this protein a transmembrane protein? • Hydrophobic alpha helices? • Typically 10-25 aa long http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2003/Bennett/protein1.htm

10. http://phobius.sbc.su.se/ • Transmembrane helix and signal peptide prediction

11. Signal (leader) peptides • Basic amino acids (+ charge) at amino terminus • Followed by hydrophobic amino acids • Trigger factor or SRP bind these

12. Compare “like” periplasmic binding proteins across species • BLAST 2GBP to find 10 examples of glucose/galactose binding protein found in 10 different bacteria

13. Compare like periplasmic binding proteins across species • Multi-sequence and multi-structure alignment • Use Muscle (multisequence) • VMD (multistructure)

14. Compare like periplasmic binding proteins within species • Use Muscle (multisequence) to align

15. Compare like periplasmic binding proteins within species • Use Weblogo (http://weblogo.berkeley.edu) to create a cool probability graph of the alignment.

16. Compare like periplasmic binding proteins within species • Use IMG (http://img.jgi.doe.go) to look at conservation across gene ortholog neighborhoods

17. Compare like periplasmic binding proteins within species • Use consurf server (http://consurf.tau.ac.il) to find conserved regions on the structure.

18. Compare unlike periplasmic binding proteins within species • Use KEGG (http://www.kegg.jp)

19. Compare unlike periplasmic binding proteins within species • Get 10 different periplasmic binding proteins from a single species • Repeat MUSCLE, Weblogo, and Consurf • Use pdb.org tools to pairwise align structures

20. Molecular Dynamics SimulationsUsing NAMD • Simulate the same protein at various temperatures • Compare the RMSD motion • Simulate different binding proteins using steering to find low energy configurations • Apo and holo structures • How do these align? • Using docking algorithms to look at binding protein recognition with trans-membrane protein

21. OLLU Introductory Biology Course (Sara): Learning Objectives Have students understand this

22. The plan: • Sara creates data set of ABC transporters (both exporters and importers) in T.cru • Explain basic structure and function of ABC transporter to students • Assign student groups each a set of 2-3 genes encoding a predicted set of ABC transporter subunits • Students use IMG-ACT to verify automated annotation call and clarify which subunit each gene likely encodes • Use similarity searching module (BLAST especially) • Cellular localization module (ID signal sequence, TM domains)

23. Undergraduate Research: Learning Objectives (Terry) • Relationship between the gene and the biochemical pathway • Inorganic transporters differ from organic transporters.

24. Undergraduate Research Projects/ Biochemistry Course • Tie Pathway to Gene • Investigate transporter relationship to biochemical pathway

25. BLAST using putative nitrate transporter as query against PDB for possible structures Result:

26. Took sequence from crystal structure and performed BLAST against Tcru genome

27. Future Directions • Extending divergence question to other organisms • Finding transporters that are tied to pathways • Finding potential Bacteriocidins • Investigating thermodynamic features that describe chemical transportation (Nernst Equation)