1. 1.Oxygen-Binding Proteins 1a. Globins 1b. Hemerythrins 1c. Hemocyanins 2.Protein catalysis (PIMT) 1a. Drosophila PIMT 1a. Pombe PIMT Relationship between protein structure, dynamics, and function.

2. Cannon 5.0 megapixel digital camera Cannon 2.0 megapixel digital camera

3. Advanced Photon Source at the Argonne National Lab

4. 1.Hydrogen atoms and hydrogen bonding. 2.Alternate conformations and sub-states. 3.Ligand geometry and Function. High-resolution Structure-Function Analysis of Oxygen-binding Proteins

5. Chironomus thummi thummi (CTT) Hemoglobin 1. Monomeric Hb 2. Bohr effect 3. Its crystals diffracts to ??? B10 G8 CD1 E11

6. 1.Obtain an unbiased view of ligand-heme stereochemistry 2.Define the Bohr effect by observing the positions of hydrogen atoms at different concentrations of hydrogen ions. 3.Define the protein dynamics of the hemoglobin within the crystal 4. Relate the proximal and distal contributions to ligand geometry. 5.Relate ligand geometry directly with protein function. Goals of the Chironomus project

7. 1.4 helix bundle 2.Coordinates with 2 Fe atoms 3.Binds a single oxygen molecule. 4.Diffracts to 1.3/1.7 Å Myohemerythrin from Themiste Zostericola (TZ) 73 106 54 25 111 58

8. 1. Define the geometry of ligand-hemerythrin complexes with atomic resolution crystallography. 2.Use site-directed mutagenesis to determine the chemical environment that is important for ligand binding (ligand stabilization and the limitation of the access of the binding site to the ligand). 3.Define the role that the coordinating residues have on the reactivity of the non-heme iron. 4.Map out the ligand migration pathways with site-directed mutagenesis and time-resolved crystallography.

9. Pictures vs. Movies

10. Time-resolved Macromolecular Crystallography 1.Follow ligand migration through the protein matrix of CTT Hb and TZ myohemerythrin. 2.Follow the ligand-induced changes that occur following ligand release. 3.Compare these results with values calculated from md simulations.

11. Determine the allosteric properties of Sea Cucumber Hb 1.Monomeric or dimeric 2.Cooperatively bind oxygen (n=1.4) 3.Similar assembly as Scapharca HbI 4.Different Mechanism of Allostery Caudina arenicola HbD

12. 1.Determine the residues that contribute to the allosteric properties of this protein. 2.Determine the routes ligands follow to enter and exit the active site (heme pocket). (Does assembly alter how ligand migration?) 3.Define the intermediates that are formed during the allosteric transition. (Are there common themes when compared to Scapharca HbI?) 4.How is the mechanism of allostery exhibited by sea cucumber Hb to other allosteric molecules? Sea cucumber Hb Goals

13. IsoAsp Formation and Protein Repair Asp IsoAsp

14. SN2SN2 Rearrangement AdoMet AdoHcy IsoAspMethylated IsoAsp Asp Reaction catalyzed by PIMT

15. N C Drosophila PIMT Structure 1.Monomeric protein. 2.SAM dependent. 3.Dynamics are important for substrate binding/exchange.

16. Human Fly N C Human vs. Fly PIMT Open and closed Conformations? PIMT over expression extends fly life spans. PIMT RNAi knockdowns ???

17. AdoHcy Tyr 218 Ser 60 N C Peptide substrate Model Methyl in AdoMet Substrate Geometry in Fly PIMT Catalytic Power 1.Solvent exclusion 2.Aprotic environment 3.Orientation of Nucleophile 4.Correct alignment of Charge

18. 1.Understand the Chemistry of PIMT. 2.Understand the dynamics of PIMT. 3.Define the targets of PIMT. 4.Understand the role of PIMT in each of this model organism. Fly PIMT Goals In collaboration with Clare O’connor at Boston College

19. Expression of PIMT in Pombe http://www.sanger.ac.uk/perl/ SPGE/geexview?q=SPAC869.08 Spore FormationMeiosis PIMT deletion mutants significantly reduces spore viability. PIMT is being cloned.

20. Pombe PIMT Goals 1.Structural determination of PIMT. 2.Enzymatic characterization of PIMT kinetics, substrate binding 3.Site directed mutagenesis 4.Replacing wild type copies with mutant proteins within Pombe.

21. Results of Phylogenetic Analysis 1.No other proteins co-vary with PIMT 2.The presence of PIMT in some related species co-varied with the absence of three chaperones (DnaJ, DnaK, and GrpE). 3.This implies, that in these species, PIMT might aid protein folding. 4.There are a few PIMT sequences which are fused to a second domain.

22. Expression Patterns of these Genes Click gene names for more options C869.08 (PIMT) and C869.07c ( putative alpha-galactosidase ) : 20 fold increase in RNA expression C869.06 ( HHE domain, S. coelicolor SC9H11.25c) and C869.09 ( N. crassa conidation protein 6) : 300 fold increase in RNA expression Spore formation Meiosis

23. Grow Crystals Mount or Freeze Crystals Collect Data Index Image Integrate spots Merge Data Determine phases Calculate electron density Build model Protein Crystallography

24. Mounting Scapharca Crystals for freezing experiments. 1.crystals are soaked in Paratone-N oil. 2.Crystals are placed into loops attached to pins 3.Pins are placed into nitrogen stream and then centered in the beam. N2N2