1. Oxygen-Binding Proteins

1a. Globins1b. Hemerythrins1c. Hemocyanins

2. Protein catalysis (PIMT)

1a. Drosophila PIMT1a. Pombe PIMT

Relationship between protein structure, dynamics, and function.

                   

     

                   

     

Cannon 5.0 megapixel digital camera

Cannon 2.0 megapixel digital camera

Advanced Photon Source at the Argonne National Lab

1. Hydrogen atoms and hydrogen bonding.

2. Alternate conformations and sub-states.

3. Ligand geometry and Function.

High-resolution Structure-Function Analysisof Oxygen-binding Proteins

Chironomus thummi thummi (CTT) Hemoglobin

1. Monomeric Hb

2. Bohr effect

3. Its crystals diffracts to ???

B10

G8 CD1E11

1. Obtain an unbiased view of ligand-hemestereochemistry

2. Define the Bohr effect by observing thepositions 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 contributionsto ligand geometry.

5. Relate ligand geometry directly withprotein function.

Goals of the Chironomus project

1. 4 helix bundle2. Coordinates with 2

Fe atoms3. Binds a single oxygen

molecule.4. Diffracts to 1.3/1.7 Å

Myohemerythrin from Themiste Zostericola (TZ)

73

106

54

25

111

58

1. Define the geometry of ligand-hemerythrincomplexes with atomic resolution crystallography.

2. Use site-directed mutagenesis to determinethe chemical environment that is important for ligand binding (ligand stabilization andthe 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 withsite-directed mutagenesis and time-resolvedcrystallography.

Pictures vs. Movies

Time-resolved Macromolecular Crystallography

1. Follow ligand migration through the protein matrix of CTT Hb and TZmyohemerythrin.

2. Follow the ligand-induced changes that occur following ligand release.

3. Compare these results with values calculated from md simulations.

Determine the allosteric properties ofSea Cucumber Hb

1. Monomeric or dimeric2. Cooperatively bind oxygen (n=1.4)3. Similar assembly as Scapharca HbI4. Different Mechanism of Allostery

Caudina arenicola HbD

1. Determine the residues that contributeto 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 formedduring the allosteric transition.

(Are there common themes when compared to Scapharca HbI?)

4. How is the mechanism of allostery exhibitedby sea cucumber Hb to other allosteric molecules?

Sea cucumber Hb Goals

O

N

OO-

O

N

OO-

IsoAsp Formation and Protein Repair

Asp IsoAsp

MeMe

NO

OO

O

N

OO-

O

N

OO

S

Me

S+

SN2

Rearrangement

AdoMet AdoHcy

IsoAsp Methylated IsoAsp

Asp

Reaction catalyzed by PIMT

O

N

OO-

O

N

OO-

N

C

Drosophila PIMT Structure

1. Monomeric protein.

2. SAM dependent.

3. Dynamics are important for substratebinding/exchange.

Human

Fly

N

C

Human vs. Fly PIMT

Open and closed Conformations?

PIMT over expression extends fly life spans.PIMT RNAi knockdowns ???

AdoHcy

Tyr 218

Ser 60

N

CPeptide substrateModel

Methyl in AdoMet

Substrate Geometry in Fly PIMT

Catalytic Power

1. Solvent exclusion2. Aprotic environment3. Orientation of Nucleophile4. Correct alignment of Charge

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 GoalsIn collaboration with

Clare O’connor at Boston College

                    

Expression of PIMT in Pombehttp://www.sanger.ac.uk/perl/SPGE/geexview?q=SPAC869.08

Spore Formation

Meiosis

PIMT deletion mutants significantly reducesspore viability.

PIMT is being cloned.

Pombe PIMT Goals

1. Structural determination of PIMT.

2. Enzymatic characterization of PIMTkinetics, substrate binding

3. Site directed mutagenesis

4. Replacing wild type copies with mutantproteins within Pombe.

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 threechaperones (DnaJ, DnaK, and GrpE).

3. This implies, that in these species, PIMT mightaid protein folding.

4. There are a few PIMT sequences which arefused to a second domain.

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 formationMeiosis

Grow CrystalsMount or Freeze CrystalsCollect Data

Index ImageIntegrate spotsMerge Data Determine phasesCalculate electron densityBuild model

Protein Crystallography

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.

N2