STRUCTURE OF PROTEIN

Definition of Proteins:

Large molecules composed of one or more chains of amino acids in a specific order determined by the base sequence of nucleotides in the DNA coding for the protein.

Proteins are required for the structure, function, and regulation of the body's cells, tissues, and organs. Each protein has unique functions. Proteins are essential components of muscles, skin, bones and the body as a whole.

Examples of proteins include whole classes of important molecules, among them enzymes, hormones, and antibodies.

Structure of protein: In order to comprehend denaturation, it is crucial to understand the structure of proteins. Proteins are composed of amino acids. Scientists describe the structure of proteins in Four levels. The four levels are primary, secondary, tertiary, and quaternary.They are…….

1. primary structure

2. Secondary structure

3. Tertiary structure

4. Quaternary structure

Protein Primary Structure:

Proteins are polymers of amino acids. Each amino acid has the same fundamental structure, differing only in the side-chain, designated the R-group. The carbon atom to which the amino group, carboxyl group, and side chain (R-group) are attached is the alpha carbon.

The primary structure of a protein is the sequence of amino acids that form the protein. Every protein has a unique sequence for the amino acids. If the amino acids are out of order the protein will not function properly

Secondary structure :

Segments of polypeptides often fold locally into stable structures that include -helices and -pleated sheets.

- Helix. In an -helix (alpha helix), the polypeptide backbone follows a helical path. There are 3.6 amino

acid residues per turn of the helix. Some protein domains assume other helical structures, but the -helix is most common.

An -helix is stabilized by hydrogen bonds between backbone amino and carbonyl groups and those in the next turn of the helix, represented as NH……O=C.  The hydrogen and oxygen atoms are attracted to one another because the H atom carries a partial positive charge and the O atom carries a partial negative charge, due to unequal sharing of electrons in N-H and O=C bonds. Diagram p. 224 of Voet & Voet text

sheet (beta sheet): Another common secondary structure is the sheet (beta sheet). In a sheet, strands of protein lie adjacent to one another, interacting laterally via H bonds between backbone carbonyl oxygen and amino H atoms. The strands may be parallel (N-termini of both strands at the same end) or antiparallel. See diagrams p. 228-229 of Voet & Voet.Because of the tetrahedral nature of carbon bonds, a -sheet is puckered, leading to the designation pleated sheet. The secondary structure of a protein describes regular repeated patterns of initial folding that are found in the protein. There are two types of folding at this level, known as alpha helices and beta sheets. An alpha helix is a spiral coil shape that is formed by hydrogen bonds. Beta sheets are formed when two amino acid chains align and form a flat or wrinkled area pattern.

Tertiary structure :

Refers to the complete three dimensional folding of a protein. Stabilization of a protein's tertiary structure may involve interactions between amino acids located far apart along the primary sequence. These may include:

Weak interactions such as hydrogen bonds and Van der Waals interactions. Ionic bonds involving negatively charged and positively charged amino acid side-chain groups. Disulfide bonds, covalent linkages that may form as the thiol groups of two cysteine residues are oxidized

to a disulfide: 2 R-SH � R-S-S-R.

Interactions with the aqueous solvent, known as the hydrophobic effect results in residues with non-polar side-chains typically being buried in the interior of a protein. Conversely, polar amino acid side-chains tend to on the surface of a protein where they are exposed to the aqueous milieu. There are, however, many exceptions in which polar residues are buried or non-polar residues exposed on the surface of a protein. Such atypical locations might be stabilized, e.g., by interaction of amino acid side-chains with enzyme prosthetic groups or other ligands, by interactions between amino acid side-chains, or by association of proteins with lipid membranes, etc.

The tertiary structure of a protein describes the shape of the protein when all the amino acids, alpha helices, and beta sheets are folded into a specific structure

Quaternary structure:

refers to the regular association of two or more polypeptide chains to form a complex. A multi-subunit protein may be composed of two or more identical polypeptides, or it may include different polypeptides. Quaternary structure tends to be stabilized mainly by weak interactions between residues exposed on surfaces polypeptides within a complex.

The quaternary structure describes a protein that is composed of two folded amino acid chain subunits that come together to form one final protein

TAQ POLYMERASE

Taq polymerase:

Taq polymerase is a heat stable enzyme used in the polymerase chain reaction (PCR) to amplify segments of DNA in the lab. It was discovered in the heat-loving bacterium Thermus aquaticus, and without it, we couldn't amplify DNA.

Function of Taq Polymerase:

1. Removing the need for adding a new enzyme during thermocycling. 2. An enzyme able to withstand the protein-denaturing conditions (high temperature) required during PCR.

Uses of taq Polrmerase:

● Taq Polymerase can be a substitute to be used in PCR.● Taq Polymerase can also be used for sequencing DNA. At times, sequencing at high temperatures helps to eliminate certain sequencing artefacts, and Taq is the ideal enzyme to use under these conditions

The cause for using taq polymerase in PCR:

It is a thermostable (Temperature stable) enzyme for PCR. It removed the need for adding a new enzyme during thermocycling. Normally, you would have to replace the enzyme because during PCR, you reach about 90C and that denatures both the DNA and the enzyme. The discovery of Taq removed this need to replace enzyme, as it allows the strands of DNA to separate and act as templates in the next round of amplification without denaturing like other enzymes.

DENATURATION OF PROTEIN

Denaturation of proteins:

Denaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent (e.g., alcohol or chloroform), or heat.

If proteins in a living cell are denatured, this results in disruption of cell activity and possibly cell death. Denatured proteins can exhibit a wide range of characteristics, from loss of solubility to communal aggregation.

Example of Denaturing in Proteins:

A classic example of denaturing in proteins comes from egg whites, which are largely egg albumins in water. Fresh from the eggs, egg whites are transparent and liquid. Cooking the thermally unstable whites turns them opaque, forming an interconnected solid mass. The same transformation can be effected with a denaturing chemical. Pouring egg whites into a beaker of acetone will also turn egg whites translucent and solid. Denaturing egg whites is irreversible

Mechanism of denaturation of proteins:

Unfolding of mesophilic proteins occurs both at temperatures higher and lower than room temperature: the high temperature transition is generally referred to as “heat denaturation” whereas that at lower temperatures is known as “cold denaturation”. We have recently identified a protein, Yfh1, whose cold denaturation occurs at accessible temperatures close to 0°C and under physiological conditions at pH 7; that is, without the need to add denaturants. The first instance in which this system was used in a general sense to study the stability of proteins was a study on the influence of alcohols at low concentrations. Measuring both thermal denaturations, and hence the stability curve, in the presence of trifluoroethanol, ethanol and methanol, we observed an extended temperature range of protein stability. We suggest that alcohols, at low concentration and physiological pH, stabilize proteins by greatly widening the range of temperatures over which the protein is stable. A second important application is illustrated by titin I28, the second case of a protein undergoing unbiased cold denaturation. The thermal stability of this protein cannot be determined by increasing the temperature because aggregation competes with unfolding.

The causes of protein denaturation:

1. Denaturation occurs when a protein is exposed to an extreme environment, such as a high level of salt, high temperature, and/or high level of acidity. Because of these extreme conditions, the function of the protein alters due to deformities along their bonds and can be permanent or temporary based on several factors such as duration of exposure or exactly how "extreme" an extreme condition was. For example, some proteins are able to withstand exposure to extreme heat for several minutes and still retain original function and work properly with minimal deformities. Therefore, depending on several factors such as time and what condition of extreme exposure it is, denaturation can be both permanent and temporary in proteins.

2. Denaturation occurs because the bonding interactions responsible for the secondary structure (hydrogen bonds to amides) and tertiary structure are disrupted. In tertiary structure there are four types of bonding interactions between "side chains" including: hydrogen bonding, salt bridges, disulfide bonds, and non-polar hydrophobic interactions. This may be disrupted. Therefore, a variety of reagents and conditions can cause denaturation. The most common observation in the denaturation process is the precipitation or coagulation of the protein.

Fig: Causes or Denaturation of proteins

Denaturing Agents:The most common factors that denature proteins includes:

1. Changes in temperature:

Heat can be used to disrupt hydrogen bonds and non-polar hydrophobic interactions. This occurs because heat increases the kinetic energy and causes the molecules to vibrate so rapidly and violently that the bonds are disrupted.

Example:

The proteins in eggs denature and coagulate during cooking. Other foods are cooked to denature the proteins to make it easier for enzymes to digest them.Medical supplies and instruments are sterilized by heating to denature proteins in bacteria and thus destroy the bacteria.

2. Changes in pH :

Most proteins at physiological pH are above their isoelectric points and have a net negative charge. When the pH is adjusted to the isoelectric point of the protein, its net charge will be zero. Charge repulsions of similar molecules will be at minimum and many proteins will precipitate. Even for proteins that remain in solution at their isoelectric points, this is usually the pH of minimum solubility.If the pH is lowered far below the isoelectric point, the protein will lose its negative and contain only positive charges. The like charges will repel each other and prevent the protein from aggregating as readily. In areas of large charge density, the intramolecular repulsion may be great enough to cause unfolding of the protein. This will have an effect similar to that of mild heat treatment on the protein structure. In some cases the unfolding may be extensive enough to expose hydrophobic groups and cause irreversible aggregation. Until this occurs such unfolding will be largely reversible.

3. Heavy Metal Salts:

Heavy metal salts act to denature proteins in much the same manner as acids and bases. Heavy metal salts usually contain Hg+2, Pb+2, Ag+1 Tl+1, Cd+2 and other metals with high atomic weights. Since salts are ionic they disrupt salt bridges in proteins. The reaction of a heavy metal salt with a protein usually leads to an insoluble metal protein salt.

This reaction is used for its disinfectant properties in external applications. For example AgNO3 is used to prevent gonorrhea infections in the eyes of new born infants. Silver nitrate is also used in the treatment of nose and throat infections, as well as to cauterize wounds.

Mercury salts administered as Mercurochrome or Merthiolate have similar properties in preventing infections in wounds.

This same reaction is used in reverse in cases of acute heavy metal poisoning. In such a situation, a person may have swallowed a significant quantity of a heavy metal salt. As an antidote, a protein such as milk or egg whites may be administered to precipitate the poisonous salt. Then an emetic is given to induce vomiting so that the precipitated metal protein is discharged from the body.

4. Reducing Agents Disrupt Disulfide Bonds:

Disulfide bonds are formed by oxidation of the sulfhydryl groups on cysteine. Review reaction. Different protein chains or loops within a single chain are held together by the strong covalent disulfide bonds. Both of these examples are exhibited by the insulin in the graphic on the left.

If oxidizing agents cause the formation of a disulfide bond, then reducing agents, of course, act on any disulfide bonds to split it apart. Reducing agents add hydrogen atoms to make the thiol group, -SH. The reaction is:

Fig: Reducing agents Disrupts disulfide bond

5. Detergents:

Detergents are amphiphilic molecules (both hydrophobic and hydrophilic parts).

Disrupt hydrophobic interactions:

o Hydrophobic parts of the detergent associate with the hydrophobic parts of the protein (coating with detergent molecules)

o Hydrophilic ends of the detergent molecules interact favorably with water (nonpolar parts of the protein become coated with polar groups that allow their association with water)

o Hydrophobic parts of the protein no longer need to associate with each other

o Dissociation of the non-polar R groups can lead to unfolding of the protein chain (same effect as in nonpolar solvents).

6. Agitation:

Whipping action stretches the polypeptide chain until the bonds break.

Example: Whipping of cream or egg whites

7. Alcohol Disrupts Hydrogen Bonding:

Hydrogen bonding occurs between amide groups in the secondary protein structure. Hydrogen bonding between "side chains" occurs in tertiary protein structure in a variety of amino acid combinations. All of these are disrupted by the addition of another alcohol.

A 70% alcohol solution is used as a disinfectant on the skin. This concentration of alcohol is able to penetrate the bacterial cell wall and denature the proteins and enzymes inside of the cell. A 95% alcohol solution merely coagulates the protein on the outside of the cell wall and prevents any alcohol from entering the cell. Alcohol denatures proteins by disrupting the side chain intramolecular hydrogen bonding. New hydrogen bonds are formed instead between the new alcohol molecule and the protein side chains.

In the prion protein, tyr 128 is hydrogen bonded to asp 178, which cause one part of the chain to be bonding with a part some distance away. After denaturation, the graphic show substantial structural changes.

Fig: Denaturation by alcohol

8. Denaturation by Acids and Bases:

Salt bridges result from the neutralization of an acid and amine on side chains. The final interaction is ionic between the positive ammonium group and the negative acid group. Any combination of the various acidic or amine amino acid side chains will have this effect.

As might be expected, acids and bases disrupt salt bridges held together by ionic charges. A type of double replacement reaction occurs where the positive and negative ions in the salt change partners with the positive and negative ions in the new acid or base added. This reaction occurs in the digestive system, when the acidic gastric juices cause the curdling (coagulating) of milk.

The denaturation reaction on the salt bridge by the addition of an acid results in a further straightening effect on the protein chain as shown in the graphic on the left.

Fig: Denaturation by acid and bases