Protein structure

Protein structure basically depends on arranged of various amino acid residues in three-dimensional way. Proteins are macro-molecules and have four different levels of structures – Primary, Secondary, Tertiary and Quaternary.

1) Primary structure: It represent the linear amino acid structure

2) Secondary structure: Local folding of amino acids in primary structure

3) Tertiary structure: Global folding of peptide strand

4) Quaternary structure: Interactions between two subunits and leads to binding of two subunits form the functional proteins

Primary structure of protein:

Protein Primary structure represents the linear sequence (order) of amino acid units in a protein. This structure stabilized by covalent peptide bonds which exhibit metastability to proteins and show higher level of stability even when heated at 1800C.

This is the final structural level of protein that remains intact when a protein is subjected to complete denaturation. It means, protein consisting of any type of structure (Secondary, Tertiary or Quaternary) finally it reaches primary structural level if you do complete denaturation.

The primary structure shows a distinct feature such as polarity, because one end of the protein contains free α-amino group and other end of the protein contains free carboxylic group.  Based on this polarity the primary structure is represented as N- terminus (α-amino group side) and C- terminus (carboxylic end). This structure determines the all other structural levels of proteins. The amino acid sequence of this structure also determines the codon sequence in the gene corresponding to particular protein.

Any alterations in codon sequence of gene can affect the primary structure of protein by changing the amino acid sequence.  These changes contribute functional changes of protein and resulted in to emerging the harmful effects in the body.


Secondary structure of protein:

In the process of protein synthesis, the word “Secondary structure” correspond to the local folding of the protein at primary structure level. In primary structure, regions that are closely present come together to form week interactions by hydrogen bonding and form secondary structure of protein.

It is the only structural level in which covalent interactions are not present but hydrogen bonds can form the noncovalent interactions in local folding.

The secondary structure of protein is very important to fulfill the functional characteristic of the protein.  The secondary structure of protein solely stabilized by hydrogen bonds.

The hydrogen bonding will occur mainly between carboxyl oxygen of one peptide bond and the imino hydrogen of another peptide bond.

The main purpose of the formation secondary structure is to reduce the polarity associated with peptide bond so that higher order structure can be generated.

CD spectroscopy can be used to study the secondary of the protein and ramachandran plot can be used to determine the feasibility of occurrence of secondary structure.

α-helix was the first every secondary structure proposed by Linus pauling in 1951. The β- plated sheet was the regular secondary structure proposed by Linus pauling and Robert corey in 1952. Both the structures, α-helix and β- plated sheet were published in general of biological chemistry.

These two forms show the regular secondary structures known for proteins. The common irregular secondary structure is the random coil form.


Helical formation of protein

The amino acids in the protein-structures arranged in linear passion. However, in biological conditions sequences of amino acids will fold regularly or irregularly by molecular attractions to give suitable structure to the protein to become metabolically active.


Types of regular right handed α-helix

Basically, three types of regular right-handed helical structures have been studied such as α-helix, 310 helix and π helix.


Alpha helix form:

Involves intra chain hydrogen bonding and most common α-helix is right handed and contains 3.6 amino acid residues per turn. The distance between adjacent amino acid residues is known as Helical raise and the distance will be 1.5Å or 15nm and helical pitch of 5.4 Å (0.54nm).

                Helical pitch = (Helical rise per residue × No of residues in a loop).

For every amino acid residue, the helix turns by 1000C. The helical repeat occurs for every 5 turns or 18 amino acids that means the same kind of amino acids are repeated for every 5 turns.

In the regular right handed α-helix, we can find the hydrogen bonded loop, it will be formed by hydrogen bonding between carboxyl oxygen of Ath amino acid and imino hydrogen of A+ 4th amino acid.

Every hydrogen bonded loop contains 13 atoms therefore this regular right handed α-helix is represented as 3.613.

The aromatic amino acid proline strongly de-stabilizes the regular α-helix because imino nitrogen of proline lacks hydrogen atom for formation of hydrogen bond, so un-bonding nature of proline distrub the α-helix formation. if proline appeared in the peptide sequence then it can form the kink in protein. In some cases, by chance if proline presence then it can completely collapse the functional property of the protein.

Glycine is the simple and basic structure of amino acid and strongly de-stabilizes the α-helix because it contains only hydrogen atom as highly flexible side chain, hence it causes immediate disturbance in regular turns in polypeptide. Therefore, this amino acid known as hinge amino acid.

The amino acids such as Arginine and Aspertate contains high polarity in their side chains, hence disturb the regular α-helix.

Hydrophobic amino acids such as Alanine, valine, leucine and methionine etc are the most suitable for the formation of regular α-helix.

The best examples for α-helix contained in their protein structure are α-keratin, myoglobin and hemoglobin.



This helix contains three amino acids per turn and ten atoms per hydrogen bonded loop.

In 310 helices the hydrogen bond can be formed between carbonyl oxygen of ith of residue and imino hydrogen of i+3rd amino acid residue (H bond pattern between i and i+3). This loop contains 3 residues per turn, consecutive amino acids make an angle of 120° around the helical axis, a helical rise per amino acid of 2 Å, and a helical pitch of 5.8-6 Å (helical rise per residue × No of residues in a loop).


pi helix 

pi helix or (πhelix) is another type of secondary structure present in some proteins. 15% of known protein structures contain these short pihelices. The biology scientists were believed that formation of pi helix was because of evolutionary adaptations derived by a single amino acid insertion into an α-helix.

π helix or 4.416 helix contains 4.4 amino acids per each turn and 16 atoms in hydrogen bonded loop.

The hydrogen bonded loop can be formed by hydrogen bond between carboxyl oxygen of Ath amino acid and imino hydrogen of A+5th amino acid.


Beta plated sheet

The hydrogen bonding formation in β-plated sheet completely opposite to α-helix. In α-helix, the hydrogen bond formation will be within the same chain (intra chain bonding) but in β-plated sheet, hydrogen bond formation will be between two chains (inter chain bonding). However, in some cases bonding can be intra chain that can form the antiparallel β-plated sheet when a given chain folds back itself to form hydrogen bond.

The two chains involved in the β-plated sheet formation are called β-strands. Based on the two β-strand arrangement, the β-plated sheet has been divided in to two forms parallel and Antiparallel.


Parallel beta-plated sheet

The hydrogen bond formation in parallel β-plated sheet strictly between inter chains.

In parallel β-plated sheet, the two β-strands are arranged in same directions. It means the amino terminus of the one strand will be closed to the amino terminus of the opposite strand.

Similarly, the C- terminus of one strand will be closed to the C-terminus of another strand. However, this close proximity of similar groups in the strands develop the intrensic repulsion it resulted into reduction of the stability.

The hydrogen bonds between the chain are not compact because an amino acid in one chain form hydrogen bonds with two different amino acids in the second chain.


Antiparallel beta-plated sheet

The hydrogen bond formation in antiparallel β-plated sheet can be formed between inter chains or between intra chains.

In antiparallel β-plated sheet, the two β-strands are arranged in opposite direction. The bond formation occur between amino group of one strand and closely located carboxyl terminus of other strand.

The hydrogen bonds are highly compact because amino acid in one chain forms the hydrogen bonds with single amino acid in the second chain.

The commercially valuable insoluble protein of silk such as fibroin and sericin etc. are the best examples for the antiparallel β-plated sheet contained proteins.

Interestingly, in addition to parallel and anti antiparallel β-sheets, the mixed β-sheets also possible. It means, we can see both type of above-mentioned sheets in mixed β-sheets. Mixed β-sheets only possible when a β-sheet or protein contain three or more β-strands.


Tertiary structure of protein:

The tertiary structure corresponding to the protein folding or three-dimensional (3D) structure

Tertiary structure is next level of structure after secondary structure formation

In this structure, the functional region of the protein produced when far away separated amino acids residues are come together and form bonding.

A protein having a properly formed 3D structure is called a native protein. This native structure of the protein will have the biological activity.

In any circumstances If protein losses the 3D structure then that protein is known as denatured protein. This denatured protein completely losses the biological activity.

The tertiary structure of the protein will be formed by both covalent and noncovalent interactions. The only possible covalent interactions can be seen in disulfide bridges which form between sulfur-containing amino acids.

Noncovalent interactions can be seen in both salt bridges and hydrophobic interactions.

Salt bridges will form between negatively charged and a positively charged side chain containing amino acids, whereas hydrophobic interactions will form between hydrophobic amino acids.

Secretary and an extracellular protein contain both covalent and noncovalent interactions but intracellular proteins are stabilized by purely noncovalent interactions.

The covalent interactions of protein can be seen in the form of intra subunit or inter chain disulfide bridges. The amino acid cysteine present in the same protein molecule is the only contributor to form the disulfide bridge by the oxidation process.

The higher order tertiary structure of proteins show more stability due to the presence of disulfide bridges which are bound by covalent interactions.

Disulfide bridges are responsible for the greater stability of extracellular proteins compare to intracellular proteins, because intracellular proteins present in highly reducing environment (cytoplasm).

In cytoplasm  formation of strong disulfide brides are not possible and therefore only noncovalent interactions stabilize the structure of intracellular proteins.

Even though so many noncovalent interactions present in the protein structure, the hydrophobic noncovalent interactions only provide the driving force for protein folding.

The tendency of hydrophobic side chains of the amino acids repel from the water molecule, it leads to the gathering of all side chains at one place and forms the hydrophobic interactions. However, these are only interactions, not true bonds.

Gathering of all hydrophobic side chains at one region in a protein is called nucleation center,  these nucleation centers can be seen in different regions of the protein.

A nucleation center is a region in a protein where folding is initiated. Often such nucleation centers constitute domains. A domain is any region in a protein structure that is capable of independent folding. Domains are generated in tertiary structure.

The sulfur group-containing amino acid methionine, aromatic amino acids phenylalanine, tryptophan and branched chain amino acids such as valine, isoleucine, and leucine are considered as hydrophobic amino acids and form the hydrophobic interactions.

The salt bridges are another kind of noncovalent interactions.

The salt bridges form between the positively charged side chain of lysine or arginine amino acid and the negatively charged side chain of aspartate or glutamate amino acid.

Hydrogen bonds are the third kind of noncovalent bonds formed by side chains of imino group-amino aids aspargine and glutamine and hydroxyl group-containing amino acids serine and threonine.

The hydrogen bonds and salt bridges can be easily disrupted compared to hydrophobic interactions, therefore the conformational changes in a protein can involve the temporary loss of salt bridges and hydrogen bonds.


Quaternary structure of the protein:

Most of the protein structures are made up of the single polypeptide chain. However, some proteins are formed by more than one polypeptide chains (subunits) by interchain interactions known as quaternary structures. Subunits of the quaternary structure are sometimes identical and other times they are completely unique each other. feature

For example, dimer is the simplest form of quaternary structure, that contains two identical subunits. This type of organization can be observed in the Cro protein (DNA-binding protein) of bacteriophage λ. While, hemoglobin is tetrameric form and is another example of tertiary structure, that contains two distinct types of peptides. Two subunits one type (designated α) and two subunits of another type (designated β).

The interactions between multi-polypeptide chains form the quaternary structure more stable.

The quaternary structure of protein is only possible for multisubunit proteins and involves intersubunit interactions.

Whatever forces stabilizing the subunits of the quaternary structure are similar to the forces stabilizing the tertiary structure.

Quaternary structure of protein stabilized by both covalent and noncovalent interactions.

As noncovalent interactions, the interchain salt bridges present, this interactions form between side chains of positively and negatively charged amino acids.

Example: The extracellular protein collagen is crucial for maintaining the structural integrity of a number of tissues in the body such as blood vessels, tendones, bone, and ligaments. The salt bridges between basic amino acid, lysine and acidic amino acid, glutamate in the peptide chains of this protein stabilized the structure of collagen.

As a covalent interaction, the amino acid containing a thiol group such as cysteine residue from two different peptide chains involved in forming a higher stabilizing bond between two inter subunits. These disulfide bonds are the only covalent interactions possible in the quaternary structure.

In some cases, hydrophobic interactions, hydrogen bonds also possible noncovalent interactions and contribute the stability to protein.

Example for quaternary structure:

Insulin is extracellular protein, which has been stabilized by both covalent and noncovalent interactions.

Whereas the intracellular proteins such as hemoglobin can have only noncovalent interactions.

In case of insulin, two interchain disulfide bridges are found between chain a and b.

In the case of hemoglobin, the quaternary structure began with the formation of heterodimer of α and β subunits which are strongly held together by hydrophobic interactions that remain intact even when a conformational change occurs in hemoglobin.

Two such heterodimers come together to form the heterotetramer, in which similar kind of subunits interact through week salt bridges that can be disrupted when hemoglobin undergoes conformational changes due to the ligand binding.

By considering each subunit, hemoglobin as myoglobin the structure of hemoglobin represented as (mb-mb2).


Thermodynamics hypothesis of protein folding

Thermodynamics is a very important subject in physical science, which focus mainly on mathematical analysis of energy relationships (heat, work, temperature, and equilibrium). Macroscopically statistical mechanics explained the laws of thermodynamics. Thermodynamics applies to a wide variety of topics in biochemistry, science, and engineering, especially physical chemistry, chemical engineering and mechanical engineering.

According to the hypothesis of thermodynamics in biochemistry, the random coiled form of the protein is the most favorable conformation for any protein because it permits the highest degree of dissorderness. If any protein required to get particular structural level conformation, first it should compensate for the enthalpy of the reaction. The explanation of this concept has been clearly described below

∆G= ∆H-T∆S

∆G for a process depends on ∆H, ∆S and temperature [T]

∆G= Free energy change

∆H= enthalpy change

∆S= entropy change

Considering the above formula. If enthalpy (ΔH) is positive, the reaction is endothermic, which means heat is absorbed by the reaction because the products of the reaction having a more enthalpy than the reactants. In another case, if ΔH is negative, the reaction is exothermic, which means the overall enthalpy decrease is achieved by the generation of heat.

For example: If we discuss the enzyme reaction, the process can be two types

Endothermic reaction: If any enzyme required energy to perform the reaction, which means the final product of the reaction having more enthalpy than the substrate, hence for reaction to happen we should provide the energy.  In this process, energy or heat will be absorbed.

Exothermic reaction: If any enzyme performs the reaction without additional energy which means the final product of the reaction having less enthalpy than reactants. In this process heat or energy will be released to surroundings.


Hence, for any reaction to perform, the value of the free energy should be negative (-∆G). The proteins in random coiled form have more entropy (∆H) than any other structure of the protein. Which means, higher entropy (randomness or disorder) of the protein provides always favorable condition unless a greater negative enthalpy value can compensate for the loss in entropy. The increase in enthalpy can obtain from interactions such as hydrogen bonding in intramolecular level, van der Waals forces, hydrophobic interaction and dipolar interactions (Salt bridges). According to sheer absolute values of enthalpy, the significant increase of enthalpy is from hydrogen bonding than other interactions in protein folding. If we see the process of protein folding, the initial interaction between two peptide bonds reduces the randomness of the polypeptide strand which is mainly by the formation of a hydrogen bond.

As above mentioned, in a randomly coiled polypeptide strand the level of entropy is more and enthalpy is less compared to a higher-ordered structure (condensed). Also, the solvent present around the protein forms hydrogen bonds with the moieties in the protein backbone. Once the folding initiated, the intermolecular hydrogen bonds will be formed by a gain in structure and it should compensate both for the loss in entropy and the loss of hydrogen bonds with the solvent.

During alpha-helix formation, initially, the linear polypeptide chain forms a single hydrogen bond between carboxyl oxygen of th amino acid and imino hydrogen of i+ 4th amino acid to form one turn, it leads to initial changes in orientation of 6 torsion angles (α, β and ω) in the polypeptide chain. Formation of additional turn to this initial nucleation site requires only orientation of three torsion angles by hydrogen bonding. Thus, it is more favorable to add turns of a helix to an initial nucleation site and this is described as a cooperative effect in helix formation.

Due to the cooperative effect, the formation of further turns more likely follow the order established in the previous step. Formation of helical structure follows exactly this. The periodicity of fold-formation can arise from sequential interactions in α- helix or long-distance sheet formation in β- plated sheets.





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