PROTEIN CHEMISTRY-III

By Dr ANURAG YADAV

CONTENT:

• Globular hemeproteins.

-myoglobin

-hemoglobin

• Structure of collagen.

• Study of the Protein structure.

Globular Hemeproteins

• Group of specialized proteins that contain heme as tightly

bound prosthetic group.

• In the hemoglobin and myoglobin, heme group serve to

reversibly bind oxygen.

A. Structure of heme:

- complex of Protoporphyrin IX & ferrous iron.

- The iron is held in center of heme molecule by bonds to 4

Nitrogen of porphyrin ring.

- Heme ferrous can form another 2 bonds.

- In hemoglobin & myoglobin one of these position is

coordinated to side chain of histidine residue of globin

molecule.

Structure of Myoglobin :

1. α -helical content:

- Compact molecule with 80% of α-

helical polypeptide.

- Folded into 8 stretches.

- Labeled from A-H.

- Terminated either by Proline/ β-

bend/ loops stabilized by Hydrogen

bonds & ionic bonds.

2.Location of Polar & Non-Polar

groups:

3. Binding of heme:

- The heme group of myoglobin sits in

a crevice in the molecule

- Lined by the non-polar amino acids.

- The proximal histidine F8 binds

directly to iron , but the distal E7,

helps to stabilize the binding of

oxygen to ferrous iron.

Structure of Hemoglobin:

1. Quaternary structure of hemoglobin:

- composed of two identical dimers (αβ)1 & (αβ)2.

- Polypeptide chain within each dimer held together primarily

by the hydrophobic interaction.

- Interchain hydrophobic interaction form strong association

btwn α-subunit & β-subunit in the dimer.

- Ionic & hydrogen bonds also occur btwn members of dimer.

- The two dimers are able to move with respect to each other

- Held primarily by the polar bonds.

- Weaker interactions btwn these mobile dimers.

- The binding of the O2 to heme iron pulls the iron into the

plane of the heme.

It is of two forms:

a. T form:

- deoxy form

- the 2 αβ dimers interact through

network of ionic bonds &

hydrogen bonds that constrain

the movement of polypeptide

chains.

- it is low Oxygen-affinity form

b. R form:

- oxy form

- binding of O2 cause rupture of

some of the ionic bonds &

hydrogen bonds btwn the

dimers.

- In which the chains have more

freedom of movement.

- it is an high Oxygen-affinity form.

STRUCTURE OF COLLAGEN 1. AMINO ACID Sequence:

- Rich in Proline and Glycine ( imp in formation of triple helix).

- Proline facilitate formation of helical conformation of each α chain of helix (because its ring structure cause kink in the peptide chain).

- Glycine ( smallest aa) found every third position.

- It is repetitive part with sequence as -gly-X-Y- (X frequently is Proline, Y often is hydroxyproline).

2. Triple helix formation

3. Hydroxyproline and hydroxylycine:

- These are seen as part of collagen

- Result from hydroxylation of some

Proline& lysine after incorporation

into polypeptide chain.

- Imp in stabilizing the triple helix (

maximizes the Interchain hydrogen

bonding).

Collagen diseases:

- Ehlers-Danlos syndrome

- Osteogenesis imperfecta

Study of Protein Structure:

• The first protein to be sequenced was insulin by Sanger.

• Before studying the structure, first pure sample of the protein has to be available.

• Various proteins are extracted and purified by-

Chormatography technique

• Purity is studied by the Electrophoresis.

• Molecular weight by the mass spectroscopy.

-Ion exchange -Adsorption -Partition -Paper chromatography -Affinity -HPLC

Steps to Determining the Primary structure:

- Determining the number of polypeptide chains in protein.

- Determine amino acid composition of polypeptide chain by

complete hydrolysis.

- Identification if N- & C- terminals

- Site specific enzymes used for hydrolysis of chain into

peptides.

- Separation and purification of each peptides and then

analyzing amino acid sequence.

Dansyl chloride FDNB

Steps in Edman's degradation process

• Finger printing Method( Ingram’s Technique)

• Automatic sequencing

• study of Higher levels of Proteins structure.

- X-ray diffraction.

- nuclear magnetic resonance(NMR).