Electrostatic Potential (ESP) Measure polarization Electron Map density Electron distribution

Dipole Moment Measure bond length/angle

Measure bond strength

Organic software for 3D model

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Modelling and 3D representation

Chemistry Database

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Spectroscopic Database

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Modelling and 3D representation

Click here crystallography database.

✓ ✓

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Click here chem EdDL

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Measure polarization Electron Map density Electron distribution

Electrostatic Potential (ESP) Dipole Moment

Measure bond length/angle Measure bond strength

Organic software for 3D model

Click here download Rasmol

Click here download PyMol Click here download Jmol

Click here Chem EDDL

Click here chemical search.

Modelling and 3D representation

Quick Chemistry Database Check

Click here down Swiss PDB

Modelling and 3D representation

✓ ✓

Click here NIST data

✓ Click here download Arguslab

Click here chemaxon quick chem check

Click here download Avagrado

Click here chem EdDL

Click here for Visualization/3D sources

Click here download Marvin Sketch

Click here quick chemical check

Click here quick chemical check

Measure bond length/angle Measure number H2 bonds

Measure bond strength Protein 1, 2 , 3O structure

Presence of disulfide bond Presence alpha and beta pleated sheet

Protein Data Bank Protein database key in - PDB 4HHB

Click here Chimera tutorial

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Uses molecular modelling

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2

Chemical viewer 3D structure (Chimera)

Download PDB text file

File – fetch by ID- 4HHB

Select – residue – HEM Select – chain A – Action – Ribbon – Hide Select – chain B,C,D - Action – ribbon Hide Display only ligand Heme Tool- structural analysis - Distance Select 2 atom -by press control/shift/left click select 2 points Tool – structure analysis – create to get distance

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Check here 4HHB Chimera 1MBO Select Histidine that are close to ring Locate His F8 and E7 Make measurement

Click here download chimera

Tool – Sequence – choose sequence for 4 chains Identify amino acids of interest

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Type PDB code – 4HHB Right click – select Hetero Select - HETATM – HEM 4 Heme is display from 4 chains

Measure bond length/angle Measure number H2 bonds

Measure bond strength Protein 1, 2 , 3O structure

Presence of disulfide bond Presence alpha and beta pleated sheet

Click here J mol protein video

Chemical viewer 3D structure (Jmol)

Uses molecular modelling

1

J mol executable file

Measure distance

Select measure – distance for porphyrin ring Measure ring size/distance Fe from plane Select protein – by residue – Histidine Measure and locate His F8 Measure and locate His E7

final heme – click here

J mol executable file

1

Type 4HHB into protein data bank Look for ligand Heme

Model kit to design molecule

Click here deoxyhemoglobin chimera

2 2

3

4

3

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final product All histidine shown

Get structure from PDB and MOL

Measure bond length/angle Measure number H2 bonds

Measure bond strength Protein 1, 2 , 3O structure

Presence of disulfide bond Presence alpha and beta pleated sheet

Organic software for 3D model (Pymol)

1 1

Click here - Protein Data Bank Protein database key in - PDB 4 letter code

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Click here download PyMol

Click here Pymol video tutorial

Click file – open your download pdb file from Protein Data bank Get to command term – Type fetch 4HHB H - Hide – S - Show cartoon – C – Type by ss

Select 4 Hem – Look for 4 Hemes Select 4HHB – H – hide everything Select Heme – Show stick Look His – select and name His F8 and His E7

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Press S – sequence at bottom screen. Right click – zoom in Select HEM - hemoglobin

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Uses molecular modelling

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Select heme – right click – action – around 5A Look for His F8 and E7 around heme Make measure for distance Double click to display name of atom

Measure bond length/angle Measure number H2 bonds

Measure bond strength Protein 1, 2 , 3O structure

Presence of disulfide bond Presence alpha and beta pleated sheet

Protein Data Bank Protein database key in - PDB 4HHB

Click here Swiss PDB tutorial

1

2

Uses molecular modelling

1

2

Chemical viewer 3D structure (Swiss PDB)

Download PDB text file

File – open 4HHB pdb downloaded from databank

Window – Control panel Remove – side chain Select – Group kind – HETATM Display – stereo view Show only selected 4 Heme

Click here down Swiss PDB

Select – Group kind – Histidine Select – Residue – close to 2A Locate Histidine and make measurement

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Check for heme and Histidine only from control panel

Select Histidine that are close to ring Locate His F8 and E7 and make measurement

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Heme

Hemoglobin - 4 chain - 4 heme - 4 Fe 2+

Fe 2+

Heme (porphyrin)

Hemoglobin - 2 alpha chain - 144 amino acid - 2 beta chain - 146 amino acid

Myoglobin - 1 chain – 1 heme – 1 Fe2+

- 154 amino acids

Hemoglobin Myoglobin

Fe 2+

Heme (porphyrin)

PDB code files

Oxyhemoglobin – 1GZX, 1JY7 Deoxyhemoglobin – 1A3N, 4HHB, 2HHB, 1HBB, 1G9V, 101J Myoglobin – 4MBN, 3RGK, 5MBN Fetal hemoglobin – 1FDM Sickle cell Hemoglobin - 2HBS, 1NEJ Cytochrome (bovine) - 2B4Z Cytochrome (horse) - 1HRC Cytochrome (yeast) - 1YCC Cytochrome (human) - 3NWV

PDB file type for data analysis

1

Analyze using Chimera/Pymol/Swiss PDB/Jmol

1

Chimera Swiss PDB

Jmol Pymol

Fe in cytochrome 1 polypeptide chain

Fe 2+

Cytochrome c

Possible Research Question

Measuring using 3D modelling

Data Collection using 3D modelling

Data Collection using Database

Click here Jmol Click here PyMol

- What is the distance bet Fe and His E7 and F8, and are they the same for diff

heme found in hemoglobin/myoglobin/Cytochrome - Is His E7/F8 orientation similar for Hemoglobin, Myoglobin, Cytochrome - Is there any differences bet distance/position/orientation of porphyrin ring for

Hemoglobin, Myoglobin, Cytochrome - How is Fe2+ located, along or out of plane for Hemo/Myoglobin/Cytochrome - Is distance bet Fe and ligand N of porphyrin the same for

Hemoglobin/Myoglobin/Cytochrome - Is there any variation in terms of Fe and His E7/F8 for

Hemoglobin/Myoglobin/Cytochrome - Why His E7 and F8 are located in such a way across many different species? Is

their orientation highly conserved and why? - Similarity among cytochromes found in diff species of organism

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Structural similarity and diff bet Hemoglobin/Myoglobin and Cytochrome

Myoglobin Hemoglobin Cytochrome

Hemoglobin Chimera Pymol Jmol Swiss PDB Mean

Orientation His/Fe Similar Similar Similar Similar Similar

Bond length N - Fe 2.12A 1.90A 2.02A 2.02A 2.01A

Bond length Fe – E7 5.93A 5.80A 5.45A 5.42A 5.55A

Bond length Fe – F8 2.25A 2.05A 2.10A 2.21A 2.13A

Chimera Swiss PDB

Data source

Myoglobin Chimera Pymol Jmol Swiss PDB Mean

Orientation His/Fe Similar Similar Similar Similar Similar

Bond length N - Fe 2.02A 2.11A 2.15A 2.32 2.14A

Bond length Fe – E7 5.80A 5.71A 5.56A 5.25A 5.25A

Bond length Fe – F8 2.15A 2.25A 2.11A 2.21A 2.21A

His E7

His F8

Fe

N

Possible Research Question Data Collection using 3D modelling

Data Collection using Database

Click here Jmol Click here PyMol

Click here NCBI Click here UCSC

Click here Ensembl

Structural similarity and differences bet Hemoglobin and Myoglobin

Myoglobin hemoglobin

vs

Chimera Swiss PDB

Evaluation and Limitation using 3D modelling

Must use a variety of sources/programme to verify/validate the validity and reliability of data collected Average is computed from diff software and checked with database to confirm. Check on methodological limitation using 3D model. (MUST perform 3D Optimization to most stable form structure. Critical and skeptical of result produced by computational chemistry. Major limitation of computation, they assume non-interacting molecule. (Ideal situation, ex molecule in vacuum or isolated state) Most appropriate molecule are those whose coordinates are not theoretical but derive from experimental structural determination (using X ray diffraction) Be careful of predicted arrangement from simulation /3D model Data sources are supported using diff method/3D model/database Certain database like NIST and CRC are more reliable source Check if there is a good agreement bet CRC, diff databases and 3D model prediction before making conclusion Computation programme is always based on approximation and we cannot conclusive prove anything Reflect of validity and reliability of data Is model a true representation of reality?

- What is the distance bet Fe and His E7 and F8, and are they the same for

diff heme found in hemoglobin/myoglobin - Is His E7/F8 orientation similar for Oxy, Deoxy and Myoglobin. - Is there any differences bet distance/position/orientation of porphyrin

ring for Hemoglobin and Myoglobin - How is Fe2+ located, along or out of plane for Hemo/Myoglobin - Is the distance bet Fe and ligand N of porphyrin the same for

Hemoglobin/Myoglobin - Is structure/size of porphyrin ring same for α and β chain - Is there any variation in terms of Fe and His E7/F8 for fetal hemoglobin

and sickle cell hemoglobin - Why His E7 and F8 are located in such a way across many different

species? Are their orientation highly conserved and why ?

- Porphyrin gp of heterocyclic made of 4 pyrrole subunit - Porphyrin macrocycle has 26 (delocalized) pi electron, obey Hückel rule - It is aromatic, 4n+2 π. (Highly conjugated system)

Heme

Porphyrin Heme = Fe + porphyrins ring

Heme

Heme A Heme B Heme C

Mitochondria - cytochrome c oxidase - electron transport

O2

Heme = Fe + porphyrin ring – carry O2

Fe2+ located

Most abundant Hemoglobin and Myoglobin

Mitochondria - cytochrome c - electron transport

Fetal Hemoglobin (2α22γ2)

Human Hemoglobin (2α2 2β2)

Sickle cell Hemoglobin (2α22βS

2) Myoglobin 1 α chain

Carbaminohemoglobin Carboxyhemoglobin Oxyhemoglobin

Heme

Heme A Heme B Heme C

Mitochondria - cytochrome c oxidase - electron transport

Most abundant Hemoglobin and Myoglobin

Mitochondria - cytochrome c - electron transport

Fetal Hemoglobin (2α22γ2)

Human Hemoglobin (2α2 2β2)

Sickle cell Hemo (2α22βS

2) Myoglobin 1 α chain

Cytochrome

Heme in cytochrome, highly conjugated ring sys surrounding Fe Cytochrome - REDOX rxn – mitochondria – ATP/energy production via elec transport chain Many type cytochromes – Cyto a, b, c1, a3 Cytochrome c, an ancient protein, developed early in the evolution of life. Essential protein for energy/ATP HIGHLY CONSERVED has changed little in millions of years. Many variation – but structure remain relatively unchanged

Fe in cytochrome Fe in cytochrome Cytochrome c – heme c

Hemoglobin A - 2 alpha and 2 beta chains Hemoglobin A2 - 2 alpha and 2 delta chains Hemoglobin F - 2 alpha and 2 gamma chains Heme (porphyrin) bind to Fe2+ using 4 nitrogen atom (histidine gp) Porphyrin

- as electron-pair donor - polydentate ligand Fe form 2 additional bonds, one on each side of the heme plane. These binding sites call fifth and sixth coordination sites. This hisitidine is referred as proximal Histidine F8 The sixth coordination site bind oxygen with His E7 nearby

Deoxyhemoglobin Fe2+ - out plane Can’t fit the ring

Heme

Hemoglobin - 4 chain - 4 heme (porphyrin) - 4 Fe 2+

Fe 2+

Heme (porphyrin)

Oxyhemoglobin Fe2+ - located in plane Fit the ring

Deoxyhemoglobin

Fe2+ out plane Can’t fit the ring Out by 0.06nm

Fe2+ in plane Fit the ring

Human hemoglobin - 2 alpha chain - 144 amino acid - 2 beta chain - 146 amino acid

Fe bind to six ligand. 4 with N atom of porphyrin Fifth ligand is donated by His F8 O2 add to Fe as sixth ligand O2 tilt relative to perpendicular of heme plane

His F8 His F8 His F8

His E7 His E7

His E7

Oxyhemoglobin

Heme

Hemoglobin - 4 chain - 4 heme (porphyrin) - 4 Fe 2+

Fe 2+

Heme (porphyrin)

Hemoglobin - 2 alpha chain - 144 amino acid - 2 beta chain - 146 amino acid

Fe bind to six ligand. 4 with N atom of porphyrin Fifth ligand is donated by His F8 O2 add to Fe as sixth ligand O2 tilt relative to perpendicular of heme plane

His E7 locate over Fe, force CO to bind to Fe at an angle. This steric hinderance reduce afinity of CO in hemoglobin. O2 bind to Fe at an angle, its binding not affected by presence of His E7.

His (E7)

His (F8)

vs

Myoglobin - 1 chain – 1 heme (porphyrin) - 1 Fe2+

- 154 amino acids

Hemoglobin Myoglobin

Fe 2+

Heme (porphyrin)

His (F8)

His (E7)