The first protein to be sequenced was insulin by Sanger in 1955 (Nobel prize in 1958). Before studying the structure, first a pure sample of the protein has to be available. The proteins are extracted and purified by various chromatography techniques (ion exchange, adsorption, partition, size exclusion, affinity, HPLC). The purity of the protein thus isolated is studied by electrophoresis (agar, PAGE, iso electric focussing). Further, molecular weight is determined by mass spectroscopy or by MALDI.

A. Steps for Determining the Primary Structure

1. Determination of the number of polypeptide chains in a protein. This is ascertained by treating them with Dansyl chloride, which combines with the N-terminal amino acid (Fig. 1). The tagged polypeptide chains are subjected to complete hydrolysis by boiling with 6 N HCI at 110°C for 18–36 hours under anerobic conditions to give a mixture of amino acids. The number and nature of the dansyl amino acids can be determined and will indicate the number of polypeptide chains in the protein. For example, if there are two different poly peptide chains in a protein, two different dansyl amino acids can be identified.

Fig1. Dansyl chloride

2. Determination of the amino acid composition by complete hydrolysis of the polypeptide chains, by chromatographic separation and quantitation.

 3. Identification of N-terminal and C-terminal amino acids (Fig. 2).

Fig2. Steps in Edman's degradation process.

4. Site specific hydrolysis of the polypeptide chain using specific enzymes to get a mixture of over lapping peptides.

5. Separation and purification of each of these peptides, and then analysing the amino acid sequence of each of the small peptides, and then deciphering the sequence of the whole protein.

1-B. End Group Analysis

 i. The N-terminal amino acid has already been identified by treatment with dansyl chloride (Fig. 1). Originally Sanger used fluorodinitro benzene (FDNB, Sanger's reagent) for identification of N-terminal amino acid (Fig. 3).

Fig3. FDNB

ii. The C-terminal amino acid may be identified by Carboxypeptidase A and B.

These enzymes specifically hydrolyse and release the C-terminal amino acid from the polypeptide chain. Continued action of the enzyme would release amino acids sequentially from the C-terminal end. Carboxypeptidase A will not act if the C-terminal residue is Arginine, Proline or Lysine. Carboxypeptidase B will act only if the penultimate residue is proline.

1-C. Sequencing

 The purified individual polypeptide chains are then sequenced using Edman's degradation technique. Edman's reagent is phenyl-isothiocyanate. It forms a covalent bond to the N-terminal amino acid of any peptide chain (Fig. 2). This can be identified. The Edman's reagent would then react with the second amino acid which now has the alpha amino group. The degradation is useful in sequencing first 10-30 amino acids.

1-D. Partial Hydrolysis

For very long chain proteins, the chain is broken into many small peptides of overlapping sequences. This is done by subjecting the polypeptide chain to hydrolysis by two or more different site specific enzymes. Each of these small peptides can be purified and subjected to Edman's degradation and sequenced.

Trypsin hydrolyses peptide bonds formed by alpha carboxyl group of Lysine and Arginine.

Chymotrypsin preferentially acts on peptide bonds formed by carboxyl group of amino acids Phe, Tyr, Trp, or Leu.

Cyanogen bromide (CNBr) attacks C-side of methionine residue and breaks the peptide bond.

Each peptide is then analyzed and the whole sequence of the polypeptide is determined as if fitting in the parts of a jigsaw puzzle. The position of disulphide bonds can be determined by cleaving the native protein sample to get fragments with intact S–S bonds. These fragments are then identified.

Finger Printing Method (Ingram's technique)

This method was developed by Vernon Ingram in 1956. It helps to easily identify any qualitative abnormalities in protein structure. Here the protein is digested into many small peptides by trypsin. The mixtures of peptides are separated by chromatography (peptide mapping). The position of the peptide containing the altered amino acid is found to be different when compared with the peptide map of the normal polypeptide, e.g. beta chain of hemoglobin in HbA and HbS.

Automated Sequencing

Using the Edman's degradation technique, sequencing can be completed within a few hours by automatic sequencers.

Study of Higher Levels of Protein Structure

The higher levels of protein structure may be studied by techniques using X-ray diffraction, ultraviolet light spectroscopy, optical rotatory dispersion, circular dichroism, nuclear magnetic resonance (NMR), etc.

NMR spectroscopy measures the absorbance of radio frequency of atomic nuclei. By studying the frequency at which a particular nucleus absorbs energy, we could get an idea of the functional group available in the molecule. Two dimensional NMR permits a three dimensional representation of the protein in solution. It also helps to study the alterations in conformation of a protein during binding with another ligand.

A beam of X-ray is diffracted by the electrons around each atom and the intensity of diffracted beam is detected by a photographic plate or collected by an electronic device. This X-ray diffraction study is possible only on crystallized proteins.

Nowadays, DNA sequencing is used to determine the amino acid sequence. In this method, at first, a rough sequencing of protein is done by Edman's method. Based on this knowledge, small length oligonucleotide primers are made. These are used to amplify the appropriate gene by polymerase chain (PCR) reaction and correct DNA clone is obtained. The sequence of that part of DNA is done. Using the knowledge of the genetic code, the sequence of the encoded protein is identified.

Chemical Synthesis of Peptides

Peptides are artificially synthesized for the following purposes:

1. To check whether the sequence analysis is correct or not.

2. The primary structure of a peptide is altered by one or two amino acids, so as to determine the biologically important area or the active center.

 3. To get pure preparations for medical or diagnostic purpose. For example, HIV antibody in the blood of AIDS patients is detected by ELISA method . For this, pure antigen from HIV is to be coated in the test tubes. Preparation of enough quantity of antigen from the virus is tedious and hazardous. The best way is to synthesize the antigenic part of the protein. Commercially it is cheaper to synthesize small peptides, than isolating them from biological sources.

Emil Fischer in 1890 developed the basic mechanism to protect or activate reactive groups of amino acids. Robert Merrifield in 1961 introduced the solid phase peptide synthesis (Nobel prize, 1984). He simplified the process by adding the carboxy terminal end amino acid to insoluble polystyrene beads, so that washing and purification processes become rapid . In principle, the carboxyl group of the last amino acid is fixed on the resin; and other amino acids are added sequentially. Insulin was the first major protein chemically synthesized. In 1964, Panayotis Katsoyannis in USA and Helmut Zahn from Germany, indpendently synthesized insulin.

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مواضيع ذات صلة

Brown Adipose Tissue Promotes Thermogenesis

Hormones Regulate Fat Mobilization

Adipose Tissue is The Main Store of Triacylglycerol in The Body

Classification of Proteins

Precipitation Reactions of Proteins

Imbalance in the Rate of Triacylglycerol Formation & Export Causes Fatty Liver

The Liver Plays A Central Role in Lipid Transport & Metabolism

HDL Takes Part in Both Lipoprotein Triacylglycerol & Cholesterol Metabolism

Tertiary Structure of Proteins

Secondary Structure of Proteins

Primary Structure of Proteins

Chylomicrons & Very-low Density Lipoproteins are Rapidly Catabolized