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Primary Structure of a Protein

Proteins are bio polymers containing a large number of amino acids joined to each other by peptide linkages having three dimensional structures.

The structure of proteins is a complex one which is divided into 4 parts. They are primary, secondary, tertiary and quaternary structures. Primary structure of protein is discussed below.


Primary Structure of a Protein Definition

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The primary structure of a protein refers to the number and sequence of amino acids, the constituent units of the polypeptide chain. The main mode of linkage of the amino acids in proteins is the peptide bond which links the α-carbonyl group of one amino acid residue to the α-amino group of the other. The proteins may consist either of one or of more peptide chains.

Rigid and Planar Peptide bond

Linus Pauling and Robert Carey demonstrated that the α-Carbons of adjacent amino acids are separated by three covalent bonds, arranged Cα -C - N - Cα. They also demonstrated that the amide C-N bond in a peptide is somewhat shorter(1.32 Å or 0.132 nm) than the C-N bond in a simple amide ((1.32 Å or 0.132 nm) and that the atoms associated with the bond are coplanar. This indicated a resonance or partial sharing of two pairs of electrons between the carbonyl oxygen and the amide nitrogen.
Planer Peptide Bond

(The planar peptide bond - Note that the oxygen and hydrogen atoms are on the opposite sides of the C-N bond. This is trans configuration.)

The oxygen has a partial negative charge and the nitrogen a partial positive charge, setting up a small electric dipole. The four atoms of the peptide group (C, H, O, N) lie in a single plane, in such a way that the oxygen atom of the carbonyl group and the hydrogen atom of the amide nitrogen are trans to each other. Virtually, all peptide bonds in proteins occur in trans configuration.

From these studies, Pauling and Corey concluded that the amide C-N bonds are unable to rotate freely because of their Partial double bond character. The backbone of a polypeptide chain can thus be separated by substituted methylene groups -CH(R)-.
Polypeptide Chain

(The three bonds between sequential Cα carbons in a polypeptide chain - The N -Cα and Cα - C bonds can be rotated, with bond angles designated Φ and Ψ respectively). The rigid peptide bonds limits the number of conformations that can be assumed by a polypeptide chain.
  • However, rotation is permitted about the bond between the nitrogen and α - carbon atoms of the main chain (N - Cα) and between the α carbon and carbonyl carbon atoms (Cα - C). By convention, the degree of rotation at the N - Cα bond is called phi (Φ) and that between Cα - C bond is called psi(Ψ).
  • Again, by convention, both Φ and ψ are defined as 0o in the conformation in which the two peptide bonds connected to a single carbon are in the same plane.
  • In principle, Φ and ψ can have any value between -180o and + 180o, but many values of Φ and ψ are prohibited by steric interference between atoms in the polypeptide backbone and the amino acid side chains.

Determination of Primary Structure of Protein

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There are several levels of peptide structure. The primary structure is the amino acid sequence plus any disulphide links. With 20 amino acids as building blocks, 20 square dipeptides, 20 cubed tripeptides,and so on, are possible.

Sanger proposed a procedure to determine the primary structure of protein. Sanger's strategy can be outlined as follows.
  1. To determine the type of amino acid present and their molar ratio.
  2. To cleave the peptide into smaller fragments, and separate them. Also, with these fragments, determining the amino acid composition of the protein.
  3. Identifying the N and C terminal of the amino acid in teh original peptide and in each fragment.
  4. Organizing all the above information and arrive at a completed picture of the amino acid sequence of a protein.

Amino Acid Analysis

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The amino acid analysis of a protein involves the acid-hydrolysis of amide or the peptide bonds. This is carried out by heating the peptide in 6 molar hydrochloric acid solution for 24 hours to give a solution that contains all the amino acids. This mixture is then subjected to ion-exchange chromatography, thereby separating all the amino acids present. The separation of these amino acids is based mainly on their acid-base properties.

As and when an amino acid comes out of chromatographic chamber, they are mixed with ninhydrin and the intensity of ninhydrin color is monitored electronically. The color obtained is compared with the standards available and the amino acids are identified.

Partial Hydrolysis of Peptide

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  1. Enzymatic cleavage of the peptide bonds breaks the protein molecule into smaller fragments, unlike an acid hydrolysis, which cleaves amide bonds indiscriminately.
  2. The enzymes that catalyze the hydrolysis of peptides are called Peptidases, protease or proteolytic enzymes.
  3. Trypsin, a digestive enzyme present in the intestine, catalyzes only the hydrolysis of peptide bonds involving the carboxyl group of a lysine or arginine residue.
  4. Chymotrypsin, another digestive enzyme, is selective for peptide bonds involving the carboxyl group of amino acids with aromatic side chains (phenyl-alanine, tryrosine. tryptophan).

One group of pancreatic enzymes, known as carboxypeptidases, catalyzes only the hydrolysis of the peptide bond to the C- terminal amino acid. In addition to these, many other digestive enzymes are known and their selectivity exploited in the selective hydrolysis of peptides.

End Group Analysis

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It is very important to be able to know the end group of an amino acid sequence, which side the N- terminal is facing and which side the C- terminal is placed in.
  1. Enzymatic hydrolysis with carboxy peptidase cleaves the C- terminal, and so, we can identify teh C- terminal. For the N- terminal analysis, several chemical methods have been devised.
  2. The N- terminal amino group is free and can act as a nucleophile. The alpha amino groups of all the other amino acids are part of amide linkages, are not free, and are much less nucleophilic.
  3. Sanger's method for N- terminal residue analysis involves treating a peptide with 1- fluoro-2, 4 - dinitrobenzene, which is very reactive toward nucleophilic aromatic substitution.
Sangers Method

The amino group of the N-terminal amino acid displaces fluoride from 1-fluoro-2, 4 dinitrobenzene and gives a peptide in which the N-terminal nitrogen is labeled with a 2, 4 - dinitrophenyl group (DNP).

Labeling the N-terminal amino acid as its DNP derivative is aminly historical interest and has been replaced by other modern methods like Edman degradation and automated sequencing of peptides.

Edman Degradation

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In Edman degradation, a peptide reacts with phenyl isothiocyanate to give a phenylthiocarbamoy, (PTC) derivative. This PTC derivative is then treated with an acid in an anhydrous medium, like nitromethane saturated with HCl, to cleave the amide bond between the N-terminal amino group and the remainder of the peptide. No other peptide bonds are cleaved in this step as amide bond hydrolysis requires water.

The product of this cleavage, called thiozolone, is unstable under conditions of its formation and rearranges to a phenylthiohydantoin (PTH), which is isolated and identified by comparing it with standard samples of PTH derivatives of known amino acids. This is carried out by chromatographic or mass spectrometry method.
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