JUSTIFICACIÓN DEL ESTUDIO

Protein structure is normally described at four levels of organisation.

A. Primary Structure: Primary structure is the linear sequence of amino acids held together by peptide bonds in its peptide chain. The peptide bonds form the backbone and side chains of amino acid residues project outside the peptide backbone. The free -NH2 group of the terminal amino acid is called as N-terminal end and the free -COOH end is called as C-terminal end. It is a tradition to number the amino acids from N-terminal end as No. 1 towards the C-terminal end. Presence of specific amino acids at a specific number is very significant for a particular function of a protein. Any change in the sequence is abnormal and may affect the function and properties of protein.

B. Secondary Structure: The peptide chain thus formed assumes a three dimensional secondary structure by way

of folding or coiling consisting of a helically-coiled, zig- zag, linear or mixed form. It results from the steric relationship between amino acids located relatively near each other in the peptide chain. The linkages or bonds involved in the secondary structure formation are hydro- gen bonds and disulfide bonds.

• Hydrogen bond: These are weak, low energy non- covalent bonds sharing a single hydrogen by two electronegative atoms such as O and N. Hydrogen bonds are formed in secondary structure by sharing H-atoms between oxygen of CO and nitrogen of -NH of different peptide bonds. The hydrogen bonds in secondary structure may form either an ααααα-helix or βββββ-pleated sheet structure.

• Disulphide bonds: These are formed between two cysteine residues. They are strong, high energy covalent bonds.

1. ααααα-Helix: A peptide chain forms regular helical coils called ααααα-helix. These coils are stabilised by hydrogen bonds between carbonyl O of 1st amino and amide N of 4th amino acid residues. Thus in ααααα-helix intra chain hydrogen bonding is present. The ααααα-helices can be either right handed or left handed.Left handed ααααα-helix is less stable than right handed ααααα-helix because of the steric interference between the C = O and the side chains. Only the right handed α-helix has been found in protein structure.

Each amino acid residue advances by 0.15 nm along the helix, and 3.6 amino acid residues are present in one complete turn. The distance between two equivalent points on turn is 0.54 nm and is called a pitch.

Small or uncharged amino acid residues such as alanine, leucine and phenylalanine are often found in α-helix. More polar residues such as arginine, glutamate and serine may repel and destabilise α-helix. Proline is never found in ααααα-helix. The proteins of hair, nail, skin contain a group of proteins called keratins rich in ααααα-helical structure (Fig. 6.9).

α αα

αα-Helices May be Amphipathic

Normally on the surface of proteins, α-helices may be wholly or partially buried in the interior of a protein. The amphipathic helix, a special case in which residues switch between hydrophobic and hydrophilic about every 3 or 4 residues. It occurs where α-helices interface with both a polar and a non- polar environment.

Occurrence: Amphipathic α-helices occur in plasma lipoproteins, in certain polypeptide hormones, in certain antibiotics, human immunodeficiency virus glycoproteins, certain venoms, and calmodulin-regulated protein kinases. 2. βββββ-Pleated Sheet Structure: β-Keratins present in spider’s web, reptilian claw, fibres of silk form almost

SECTION TWO

are most common to form βββββ-pleated sheet. Proline occurs in βββββ-pleated sheet although it tends to disrupt the sheets by producing kinks. Silk fibroin, a protein of silkworm is rich in βββββ-pleated sheets (Fig. 6.10).

3. Triple Helix: Collagen is rich in proline and hydroxy- proline and cannot form ααααα-helix or βββββ-pleated sheet. It forms a triple helix. The triple helix is stabilised by both noncovalent as well as covalent bonds. Interchain hydrogen bonds between different peptide chains are formed which are almost perpendicular to the long axis of α-helix. In addition, interchain additional cross links, secondary amide bonds of peptide bonds also are responsible for triple helix.

Note: Many globular proteins have mixed secondary structure of α-helix, β-pleated sheet and non-helical, non-pleated structures called random coil.

4. Reverse Turns or βββββ-bends: Since the polypeptide chain of a globular protein changes direction two or more times when it folds, the conformations known as reverse turns or βββββ-bends are important elements of secondary structure. Reverse turns usually occur on the surfaces of globular proteins where there is little steric hindrance to resist a change in the direction of the polypeptide chain.

Types of Reverse Turns: Two major types, Type I and Type II of reverse turns, each spanning 4 amino acid residues, are particularly common. In each type the carbonyl oxygen of the first residue is hydrogen bonded to the amide hydrogen of the fourth residue, stabilising a loop of 10 atoms. Type I and Type II reverse turns differ by a 180o _{rotation of the central amide plane of} the loop. The third residue of a Type-II reverse turn can only be glycine because the side chains of all other amino acid residues are too large to fit into the restricted space. Because the five-membered ring of proline has little fully extended chain. A conformation called βββββ-pleated

sheet structure is thus formed when hydrogen bonds are formed between the carbonyl oxygens and amide hydro- gens of two or more adjacent extended polypeptide chains. Thus the hydrogen bonding in βββββ-pleated sheet structure is interchain. The structure is not absolutely planar but is slightly pleated due to the angles of bonds. The adjacent chains in β-pleated sheet structure are either parallel or antiparallel, depending on whether the amino to carbonyl peptide linkage of the chains runs in the same or opposite direction. In both parallel and antiparallel β-pleated sheet structures, the side chains are on opposite sides of the sheet. Generally glycine, serine and alanine

Fig. 6.10: (H --- O) Hydrogen bond—β-pleated sheet

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conformational mobility, it is ideally suited for the second residue in a reverse turn.

5. Super Secondary Structures: Various combinations of secondary structure, called super secondary structure, are commonly found in globular proteins.

These are:

• βββββ-ααααα-βββββ unit (Fig. 6.11) • Greek key (Fig. 6.12) • βββββ-meander (Fig. 6.13)

1. βββββ-ααααα-βββββ Unit: The β-α-β unit consists of two parallel β- pleated sheets connected by an intervening strand of α- helix (Fig. 6.11).

2. Greek Key: Another common super secondary struc- ture is called a Greek Key, a conformation that takes its

name from a design often found on classical Greek pottery (Fig. 6.12).

3. βββββ-meander: The β-meander consists of five βββββ-pleated sheets connected by reverse turns (Fig. 6.13). The β-meander contains nearly as many hydrogen bonds as an α-helix, and its common occurrence probably reflects the stability conferred by this extensive hydrogen bonding.

C. Tertiary structure: The polypeptide chain with secondary structure mentioned above may be further folded, superfolded twisted about itself forming many sizes. Such a structural conformation is called tertiary structure. It is only one such conformation which is biologically active and protein in this conformation is called as native protein. Thus the tertiary structure is constituted by steric relationship between the amino acids located far apart but brought closer by folding. The bonds responsible for interaction between groups of amino acids are as follows:

• Hydrophobic interactions: Normally occur between nonpolar side chains of amino acids such as alanine, leucine, methionine, isoleucine and phenylalanine. They constitute the major stabilising forces for tertiary structure forming a compact three-dimensional structure.

• Hydrogen bonds: Normally formed by the polar side chains of the amino acids.

• Ionic or electrostatic interactions: These are formed between oppositely charged polar side chains of amino acids, such as basic and acidic amino acids. • Van der Waal Forces: Occur between nonpolar side

chains.

• Disulfide bonds: These are the S–S bonds between -SH groups of distant cysteine residues.

D. Quaternary Structure: Many proteins are made up of only one peptide chain. However, when a protein consists of two or more peptide chains held together by non-covalent interactions or by covalent cross-links, it is referred to as the quaternary structure. The assembly is often called as oligomer and each constituent peptide chain is called as monomer or subunit. The monomers of oligomeric protein can be identical or quite different in primary, secondary or tertiary structure.

Examples: Protein with two monomers (dimer) is an enzyme called creatine phosphokinase (CPK).

Haemoglobin and lactate dehydrogenase (LDH) are tertramers consisting of four monomers. Apoferritin, an apoprotein of ferritin, an iron binding and storage protein contains 24 identical subunits. An enzyme aspartate transcarbamoylase has 72 subunits in its structure.

Fig. 6.12: Greek key

Fig. 6.11:β-α-β unit

SECTION TWO

CLINICAL ASPECT

Diseases Resulting from Altered Protein Conformation

A. Prions and Prion Diseases

Prions: Prions are infectious proteins that contain no nucleic acid. Earlier thought to be an infectious agent or a virus. This infectious protein-prions was discovered in 1982 by Stanley Prusiner.

Abnormal or pathological prions cause several fatal neurodegenerative disorders known as “transmissible spongiform encephalopathies” (TSEs) or Prion Diseases.

Types of Prions: There are two isoforms:

(a) Normal or physiologic PrP—called (PrPc) or PrP-Sen. (b) Abnormal or pathologic Prp—called PrPsc or PrP-res.

(a) Normal or physiologic PrPc: It consists of 253aa found in leucocytes and nerve cells. Gene of this PrP is located in short arm of chromosome 20. This protein is heat sensitive and protease sensitive.

(b) Abnormal or pathologic PrP: The pathologic isoform is heat resistant and protease resistant. This form is associated with TSEs or prion diseases.

Defect: The basic defect involves alteration of ααααα-helical structure into βββββ-pleated sheet.

Both PrPc and PrP-Sc have identical primary structural and post-translational modification but different tertiary and quarternary structures.

PrPc is rich in ααααα-helix but PrP-Sc consists predomi- nantly of βββββ-sheet. This structural change occurs when PrP-c interacts with the pathologic isoform Pr-Psc.

Pr-Psc serves as a template upon which the ααααα-helical structure of Pr-Pc becomes the βββββ-sheet structure charac- teristic of Pr-Psc.

Prion Diseases: May manifest themselves as genetic, infectious or sporadic disorders. The disease can occur in humans and also in animals. Prion diseases can be trans- mitted by the protein alone without involvement of DNA or RNA.

Clinically rapidly progressive dementia sets in with neurological defects and ataxia.

(a) In humans: The disease is called “Creutzfeldt-Jakob Disease” (CJD).

Other human forms of the disease are:

• Gerstmann-Straüssler-Scheinker Disease (GSSD), and • Fatal familial insomnia (FFI)—rare.

(b) In animals: It produces:

• Scrapie in sheep—From this the term ‘Pr-Psc’ derived. • Bovine spongiform encephalopathy (BSE) in cattle.

Also known as ‘Mad Cow Disease’.

Pathological Changes

Each of the above disease is characterised by spongiform changes, astrocytic gliosis and neuronal loss resulting from deposition of insoluble proteins in stable amyloid fibrils.

The protofilaments of amyloid fibrils contain pairs of β-sheets in a helical form that are continuously hydrogen bonded all along the fibrils.

Pr Psc is rich in β sheet with many hydrophobic aminoacyl side chains exposed to solvent. PrPsc molecules therefore associate strongly with one another, forming insoluble protease-resistant aggregates.

B. Alzheimer’s Disease

Refolding or misfolding of another protein endogenous to human brain tissue, βββββ-amyloid is also a prominent feature of Alzheimer’s disease found in old age.

The characteristic senile plaques and neurofibrillary bundles contain aggregates of the protein, βββββ-Amyloid, a polypeptide produced by proteolytic cleavage of a larger protein known as Amyloid precursor protein (APP).

In Alzheimer’s disease patients, levels of β-amyloid become elevated, and this protein undergoes a conformational transformation from a soluble α-helix rich state to a state rich in β-sheet and are prone to self-aggregation.

Apolipoprotein E has been implicated as a potential mediator of this conformational transformation. (For details Refer to Chapter 17).