Oxygen-Binding Proteins

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Relationship between protein structure,
dynamics, and function.

• Oxygen-Binding Proteins
• 1a. Globins
• 1b. Hemerythrins
• 1c. Hemocyanins
• Protein catalysis (PIMT)
• 1a. Drosophila PIMT
• 1a. Pombe PIMT

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Cannon 2.0 megapixel digital camera
Cannon 5.0 megapixel digital camera
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Advanced Photon Source at the Argonne National
Lab
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High-resolution Structure-Function Analysis of
Oxygen-binding Proteins

1. Hydrogen atoms and hydrogen bonding.
2. Alternate conformations and sub-states.
3. Ligand geometry and Function.

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

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

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Myohemerythrin from Themiste Zostericola (TZ)

• 4 helix bundle
• Coordinates with 2
• Fe atoms
• Binds a single oxygen
• molecule.
• 4. Diffracts to 1.3/1.7 Å

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73
54
111
25
106
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• 1. Define the geometry of ligand-hemerythrin
• complexes with atomic resolution
• crystallography.
• 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).
• Define the role that the coordinating residues
• have on the reactivity of the non-heme iron.
• Map out the ligand migration pathways with
• site-directed mutagenesis and time-resolved
• crystallography.

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Pictures vs. Movies
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Time-resolved Macromolecular Crystallography

• Follow ligand migration through the
• protein matrix of CTT Hb and TZ
• myohemerythrin.
• Follow the ligand-induced changes
• that occur following ligand release.
• Compare these results with values
• calculated from md simulations.

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Determine the allosteric properties of Sea
Cucumber Hb
Caudina arenicola HbD

• Monomeric or dimeric
• Cooperatively bind oxygen (n1.4)
• Similar assembly as Scapharca HbI
• Different Mechanism of Allostery

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Sea cucumber Hb Goals

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

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IsoAsp Formation and Protein Repair
IsoAsp
Asp
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Reaction catalyzed by PIMT
AdoMet
AdoHcy
SN2
IsoAsp
Methylated IsoAsp
Rearrangement
Asp
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Drosophila PIMT Structure

• Monomeric protein.
• 2. SAM dependent.
• 3. Dynamics are
• important for substrate
• binding/exchange.

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Human vs. Fly PIMT
N
C
Open and closed Conformations?
PIMT over expression extends fly life spans. PIMT
RNAi knockdowns ???
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Substrate Geometry in Fly PIMT
Peptide substrate Model
C
N
Tyr 218
Methyl in AdoMet
AdoHcy
Ser 60

• Catalytic Power
• Solvent exclusion
• Aprotic environment
• Orientation of Nucleophile
• Correct alignment of Charge

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Fly PIMT Goals
In collaboration with Clare Oconnor at Boston
College

• 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.

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Expression of PIMT in Pombe
                                       
http//www.sanger.ac.uk/perl/ SPGE/geexview?qSPAC
869.08
Spore Formation
Meiosis
PIMT deletion mutants significantly reduces spore
viability.
PIMT is being cloned.
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Pombe PIMT Goals

• Structural determination of PIMT.
• Enzymatic characterization of PIMT
• kinetics, substrate binding
• Site directed mutagenesis
• Replacing wild type copies with mutant
• proteins within Pombe.

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Results of Phylogenetic Analysis

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

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Expression Patterns of these Genes
Spore formation
Meiosis
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
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Protein Crystallography

Grow Crystals Mount or Freeze Crystals Collect
Data
Index Image Integrate spots Merge Data Determine
phases Calculate electron density Build model
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Mounting Scapharca Crystals for freezing
experiments.

• crystals are soaked in Paratone-N oil.
• Crystals are placed into loops attached to pins
• Pins are placed into nitrogen stream and then
• centered in the beam.

N2
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