BIOCHEMISTRY - Textbook - Ostapchenko L. I. - 2012

Chapter 5. STRUCTURE, PROPERTIES, AND FUNCTIONS OF PROTEINS

5.2. Protein Structure

5.2.1. Primary Protein Structure

Amino acid residues in a protein's peptide chain do not alternate at random; rather, they are arranged in a specific order. The linear sequence of amino acid residues in a polypeptide chain is referred to as the Introduction/19.html">Primary Structure of a protein.

The primary structure of each individual protein is encoded in a DNA region called a Gene. During Protein Synthesis, the information contained within the gene is first transcribed into mRNA, and then, using the mRNA as a template, the Primary Cell/13.html">Protein Structure is assembled on the ribosome.

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Fig. 5.3. Stages of Protein conformation formation:

structures: 1 - primary, 2 - secondary, 3 - tertiary, 4 - quaternary

Each of the 50,000 individual Proteins in The Human Body has a primary structure unique to that specific protein. All molecules of a given individual protein share the same sequence of amino acid residues, which is the primary feature distinguishing this particular protein from any other.

Methods FOR STUDYING protein primary structure. Investigating the Primary Structure of Proteins is of profound general biological and medical significance. By examining the arrangement of amino acid residues in individual proteins and correlating these findings with the Spatial Organization of the molecule, fundamental patterns in The formation of protein spatial structures can be uncovered.

Furthermore, many Genetic Disorders result from disruptions in protein Amino acid sequences. Information regarding the primary structure of both normal and mutant Proteins can be invaluable for diagnosing and predicting the progression of diseases.

Determining the primary structure of proteins involves two main stages:

✵ determining the Amino Acid Composition of the protein under study;

✵ determining the Amino Acid Sequence of the protein.

The First stage in establishing protein primary structure involves a Qualitative and quantitative Assessment of the amino acid composition of a given individual protein. It is important to note that this analysis requires a specific amount of pure protein, free from contaminants of other proteins or Peptides.

To determine The amino acid composition, all peptide bonds in the protein must be broken down. The protein under analysis is hydrolyzed in 6 mol/l HCl at a Temperature of 110 °C for 24 hours. This Treatment cleaves the peptide bonds in the protein, leaving only free Amino Acids in the hydrolyzate. Additionally, glutamine and asparagine are hydrolyzed to glutamic and aspartic acids (i.e., the amide bond in the side chain is broken and the amino group is cleaved off).

The mixture of amino acids obtained by acid Hydrolysis of Proteins is separated using a cation-exchange resin Column. Such a synthetic resin contains tightly bound negatively charged groups (e.g., sulfanilic acid residues -SO₃^-) to which Na⁺ ions are attached (Fig. 5.4).

The amino acid mixture is introduced into the cation exchanger in an acidic medium (pH 3.0), where the amino acids predominantly exist as cations, meaning they carry a positive charge. Positively charged amino acids bind to the negatively charged resin particles. The greater the net charge of an amino acid, the stronger its bond with the resin. For instance, the amino acids Lysine, Arginine, and Histidine bind most strongly to the cation exchanger, whereas aspartic and glutamic acids bind the weakest.

Fig. 5.4. Separation of amino acids by Ion-exchange Chromatography:

A - chromatographic column packed with cation-exchange resin. B - stages of amino acid separation: RP - resin particles;

1 - binding of amino acids to resin particles; 2 - release of amino acids at a specific pH and NaCl concentration

Amino acids are eluted from the column by washing them with a buffer solution of increasing Ionic strength (i.e., increasing NaCl concentration) and pH. As the pH rises, amino acids lose a proton, which decreases their positive charge and, consequently, the strength of their bond with the negatively charged resin particles.

Each amino acid emerges from the column at a specific pH and ionic strength. By collecting the solution (eluate) emerging from the bottom of the column in small portions, fractions containing individual Amino acids can be obtained.

The amount of each amino acid in a given protein is determined by heating individual amino acid fractions with ninhydrin, which forms red-violet compounds. The color intensity in the sample is proportional to the amount of the amino acid it contains. By spectrophotometrically measuring the light absorbed by the ninhydrin derivatives, the content of each amino acid in the protein hydrolysate can be determined.

The process of separation and Quantitative determination of amino acids in a protein hydrolysate is fully automated and carried out using a specialized device known as an amino acid analyzer.

The Second Stage in determining the primary structure of proteins involves establishing the amino acid sequence of the protein. Determining the N-terminal amino acid in a protein and the amino acid sequence in oligopeptides is carried out using specific Reagents.

Phenylisothiocyanate (PITC) is a reagent used to determine the N-terminal amino acid in a peptide. PITC reacts with the $\alpha$-amino and $\alpha$-carboxyl groups of free amino acids, as well as with the N-terminal amino acid in peptides:

As a result of the reaction with the N-terminal amino acid of the polypeptide, a phenylthiohydantoin derivative is formed, in which the peptide bond between the $\alpha$-carboxyl group of the N-terminal Amino Acid and the $\alpha$-amino group of the second amino acid in the peptide is destabilized. This bond is selectively hydrolyzed without damaging other peptide bonds:

Following the reaction, the PITC-AK₁ complex is isolated and identified by chromatographic methods. PITC can be reused to determine the next amino acid in the shortened peptide obtained from the previous cycle. This process of sequential ("step-by-step") Cleavage of the peptide from the N-terminus has been automated and implemented in a device called a Sequencer, which makes it possible to determine The sequence of amino acid residues in oligopeptides consisting of 10-20 amino acids.

Many peptides have a primary structure consisting of more than 100 amino acids. Since sequencers are most efficient at determining the amino acid sequence of only small peptides, large molecules are first cleaved into fragments.

By using several different cleaving agents (which can be Enzymes or chemical substances) on different samples of the purified polypeptide, overlapping fragments with known amino acid sequences can be obtained. These make it possible to reconstruct the correct order of the fragments and determine the complete amino acid sequence of the polypeptide chain.

Several different Enzymes can be used for the Specific Cleavage of peptide bonds. Most commonly, Enzymatic hydrolysis of the polypeptide is performed using the proteolytic enzyme Trypsin, which belongs to the group of digestive enzymes (produced by the Pancreas). The enzyme exhibits high Specificity; it cleaves peptide bonds formed by the carboxyl group of lysine or arginine residues:

Based on the determined number of lysine or arginine residues, the number of fragments produced during tryptic hydrolysis can be predicted. Thus, a polypeptide chain containing six arginine and lysine residues will yield seven fragments upon trypsin cleavage. The amino acid sequence is then established for each fragment.

In some cases, chemical hydrolysis is preferred over enzymatic hydrolysis. For instance, the reagent Cyanogen bromide cleaves only those peptide bonds in which the carboxyl group belongs to a Methionine residue. Knowing the number of methionine residues in the polypeptide chain makes it easy to predict the number of resulting fragments. The amino acid sequence for each fragment is subsequently determined using a sequencer.

To correctly establish the sequence of the obtained polypeptide fragments, it is necessary to generate peptides with overlapping amino acid sequences. This is achieved by treating separate samples of the given polypeptide with different reagents that cleave the protein at different sites. A sufficient number of cleavages must be performed to obtain a set of peptides that ensures the overlap of all regions required to determine the sequence of the original polypeptide:

Due to interactions between the Functional groups of their amino acids, the linear polypeptide chains of individual proteins acquire a specific spatial three-dimensional structure known as conformation. All molecules of individual proteins (i.e., those with identical primary structure) adopt the same conformation in solution. Consequently, all the information required for the formation of spatial structures is encoded within the primary structure of proteins.

Proteins exhibit two MAIN TYPES OF polypeptide chain conformation: secondary and tertiary structures.