Proteins

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Proteins

Protein Structure

1. Primary Structure

• The amino acid sequence of a


polypeptide is its primary
structure.

2. Secondary structure

• Most common types are @helix and Beta- pleated sheet which are
stabilized by H-bond between carbonyl and N-H groups.

@-Helix

• In almost all proteins, the helical twist of the helix is right handed.

• There are 3.6 amino acid residues per turn of the helix and the pitch
is 0.54 nm. Each residue from other is separated by 1.5 A° (0.15
nm).

• Intrachain H-bond forms between N-H and carbonyl group of each


amino acid are 4 residues away.
• Except for amino
acids near the
ends, all the CO
and NH forms
H-bond.

• The @-helix cannot


be formed by
mixed copolymer of
D- and L- amino
acids. L-amino acid
can form either
right or left
handed @-helices.

• The proline tends to disrupt @-helices because it lacks -NH group and
because its ring structure restricts its value near 60°

B pleated sheets

• It forms when two or more polypeptide chain line up side by side.

• The distance between adjacent amino acids along beta-strand is 3.5


A° approx, in contrast with a distance of 1.5 A° along an @-helix.

• They have H-bonds formed between polypeptide backbone N-H and


carbonyl group of adjacent strands.

• The Antiparallel -sheets are more stable than parallel because fully
B
colinear H-bonds form.
3. Tertiary Structure

• It is the 3-d (3°-structure) conformation that globular protein have


due to interactaction between the side chains in 1° structure.

• A domain is the fundamental unit of 3° structure and functional unit


of protein. Following are the interactions in 3° structure -

1. Hydrophobic interactions (non-covalent)


2. Electrostatic interactions (salt bridges)
3. Hydrogen bonds
4. van der Walls force of interaction
5. Covalent bonds - It is the inter chain disulphide bond form between
two cystein residues due to oxidation of their thiol, forming oxidized
dimer called cystine.

D
4. Quaternary Structure

• The Polypeptide subunits join to form the quaternary structure (4°).

• Non-Covalent Interactions -
1. Hydrophobic
2. Electrostatic (salt bridges)
3. Hydrogen bonds

• Covenlent Interactions -
1. Interchain disulphide bond

• Many proteins like hemoglobin (multimeric protein), are composed of 2


or more polypeptide chains and each chain called as a subunit.

• Average Molecular Weight of Std. Amino Acid = 128 Da


• Amino Acid weight during peptide formation = 110 Da (128-18) Da

• Water molecule released during peptide formation = 18 Da T

Denaturation of Proteins
• Denaturation of proteins is the loss of native conformation. It means
loss quaternary, tertiary and secondary structure of proteins.

• It includes the breaking of covalent and non-covalent bond but usually


doesn't include the breaking of peptide bonds.

• Denaturation makes complete or partial loss of protein's biological


activity.

• Urea is most commonly used denaturing agent.

• It is said that urea makes H-bonds with polar moeties of protein, by


altering the solvent environment diminishing the hydrophobic effect.
Solubilities of Proteins

1. Effect of pH

• Above and below the pI causes for increase in the -ve and +ve charge
molecule respectively and therefore creates repulsion.

• At the pI (isoelectric), the -ve and +ve charge on the protein


molecule cancel one another.

• Thus the electrostatic repulsion no longer occurs and attraction may


occur, resulting in the formation of a precipitate due to decrease in
solubility (Isoelectric precipitation).

2. Effect of Ionic strength

• The solubility of protein at low ionic strength generally increases with


increasing salt concentration. This process known as salting in.

• It occurs due to binding of salt ions to the protein ionisable groups by


decreasing the interaction between oppositely charged group on
protein molecule.

• Later water molecule forms salvation spheres around these groups.


However, when large amount of salt are added, a precipitate forms.
This process is known as salting out.

• Salting out depends on the hydrophobic nature of the protein surface.


3. Effect of Solvent

• The organic solvents such as acetone, ethanol, decreases the


dielectric constant causes the electrostatic force of attraction
between the two proteins.

• The solvents also lower the solvating power of aqueous solution.

Simple and Conjugate Proteins

• The simple proteins contain only amino acids. eg; Serum Albumin

• The conjugated proteins (holoprotein) contain simple protein and non-


protein (prosthetic) group. The proteins without prosthetic group
known as apoprotein.

IT
Fibrous and Globular Proteins

Ei
Collagen

• It is the major structural protein in the extracellular matrix. It is


the most abudant protein in vertebrates.

• They contain atleast 19 different members. A typical collagen


molecule is long, inelastic, stiff , triple stranded helical structure.

• Fundamental unit of collagen tropocollagen (300 nm), consists of


3-coiled polypeptides (.a chains).

• The chain are left handed polypeptide helices having 3.3 amino
acid residues per turn that wind around one another in characteristic
right-handed triple helix.
• The 25 different kinds of a chain forms 19 different collagen
molecules (Type - I, II, and III → 90% collagens).

Structure

• The amino acid sequence of a chain is generally tripeptide unit,


Gly-X-Y, where
X → proline, often
Y → 3- or 4-hydroxyproline or 5-hydroxylysine, often

• The glycine = approx. 1/3 of amino acid residue. And proline and
hydroxyproline causes for the rigidity of collagen.

• The hydroxylation carried out by post-translational modification of


the a chain by two enzymes, those enzymes are -
1. prolyl hydroxylase
2. lysyl hydroxylase

• Ascorbate (vitamin C) is essential for above 2 enzymes.

• The hydroxylation forms interchain H-bonds and also allows the


glycosylation of hydroxylysine residues.

• Deficiency of ascorbic acid causes scurvy, a disease that affects


collagen structure. Scurvy occurs due to impaired synthesis of
collagen due to deficiencies of prolyl and lysyl hydroxylases.
Synthesis

• The x chain are synthesized on RER's ribosomes and enter its lumen.

• In the lumen, selected

Proline and Lysine hydroxylated Hydroxyproline and Hydroxylysine

• And some hydroxylsine residues are glycosylated.

• Then 3 a chain combines (H-bond) to form tropocollagen (triple-


stranded helical) which is secreted into extracellular space to form
collagen fibrils.

I
Elastin

• A hydrophobic connective tissue protein responsible for extensibility


and elasticity.

• It is 2nd major protein in extracellular matrix, which is the main


component of elastic fibers found in ligaments, arteries and lungs.

• The tropoelastin (72 KDa) is secreted into matrix. It is rich in -

1. Non-polar amino acid


2. Proline
3. Glysine

• It is not glycosylated (no hydroxylysine) and contain hydroxyproline.

• The tropoelastin composed of 2 types of short seq. that alternate


along the polypeptide chain.

Pathway

• After secretion, the tropoelastin become highly cross-linked with each


other and form extensive network of elastin fibres.

• After secretion, the lysyl residue oxi-deaminated to aldehydes.


• These 3 aldehydes condensation with unmodified lysine forms the
desmosines (tetrafunctional cross-link).

• Once cross-linked in its mature and extracellular form, elastin is


highly insoluble and extremely stable.

Keratin

• The fibrous protein present in eukaryotes. They have 30 members.


They are classified as either
x keratin or keratin.

• The @- keratin present in many metazoans, including vertebrates. In


vertebrates they constitute almost entire dry weight of hair, wool,
nails, horns, etc.

• The @-keratin polypeptide chain is right handed and amino-acid rich.

• 2 @-keratin → heterodimer ( Two chains- 1. Acidic 2. Basic/neutral


and parallel )

• 2 Heterodimer → Protofilament ( 2 Chains joined Antiparallel and is a


tetrameric subunit )

• 2 Protofilament → Protofibril, 4- Protofibril→ Microfibril


Microfibril → Macrofibril
Myoglobin

Distal
Histidine

N
n
Proximal
Histidine

• It is a globular protein, contains a single polypeptide chain of 153


amino acid residues ( M.W - 17,800 Da) and a single heme group.

• The myoglobin consists -

1. The non-polar residues inside.


2. The polar and non-polar residues, both, outside.
• About 75% of the polypeptide chain is @-helical. There are 8 helicals.

• The iron atom of heme is directly bonded to a N-atom of Histidine -


side chain of globin.
Hemoglobin

• Hemoglobin (M.W - 65,450 Da) is an oligomeric, allosteric, conjugated


protein with 4-polypeptide chains joined by non-covalent bonds.

• The heme group it contains is porphyrin derivative.

• It is present in all vertebrates (except Artic fish) and in many


invertebrates including annelids, many arthropods and some molluscs.
• All Hb are tetramers having 7 distinct polypeptide chains.

• The variety of hemoglobin found in humans are -

1. HbA1 (92132) - Contains 2 X and 2 B chains. Makes 98% of Adult


human Hb.

2. HbA2 (
9282) - Contains 2 8 and 2 X chains. Makes 2% of adult
Hb.

Oxygen Transport

• About 98.5% of blood oxygen bound to hemoglobin in rbc and 1.5% in


plasma (which is mostly water).

• Iron in Fe2+ state binds to oxygen to form oxyhemoglobin.


• While CO and NO also binds to heme.

• The hemoglobin exists in 2 conformation state,

1. Tensed (T) - Predominant conformation of deoxyHb and stabilized by


greater no. of ionic interactions.

2. Relaxed (R) - The oxygen binds with higher affinity in this state.

• The binding of 1st oxygen molecule with deoxy state occurs weakly,
because it binds to a subunit in T state and starts a conformational
change.

• After 1st binding, transition from T to R state occurs more readily in


2nd subunit once oxygen is bound to first subunit.

• The last (4th) oxygen molecule binds to heme in a R state subunit with

higher affinity than 1st oxygen molecule.


Factors affecting the affinity of Hb for oxygen

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