Proteins are the major components of living organisms and perform a wide range of essential functions in cells.

Proteins regulate metabolic activity, catalyze biochemical reactions and maintain structural integrity of cells and organisms.

Classification of Proteins According to biological function:

Type: Example: DNA-polymerase 1.Enzymes- catalyze biological reactions Dehydrogenases Ribonuclease 2.Hormones 3.Transport protein 4.Movement proteins 5.Immune Protection proteins 6.Receptors 7.Signalling proteins regulatory function within cells 8.Structural proteins 9.Storage proteins Insulin Hemoglobin Actin and Myosin in muscles Immunoglobulins (antibodies) Hormone receptors rhodopsin Transcription & Translation Factors Collagen Keratin Egg ovalbumin milk casein

Proteins are the most abundant and diverse molecules found in living cells. How does one group of molecules perform such a diferent set of functions? The answer is found in the wide variety of possible structures for proteins.

Ribonuclease

Collagen

Hemoglobin

An incredible variety of proteins can be formed using 20 different monomers called amino acids.

The a-amino acids in peptides and proteins (excluding proline) consist of a carboxylic (-COOH) and an amino (-NH2) functional group attached to the same carbon atom. This carbon is the alpha-carbon. R-groups (radicals), that distinguish one amino acid from another, also are attached to the alpha-carbon.

Amino acid structure

Classification of amino acids

In dependence of radical (R) structure (there are aliphatic (acyclic) aromatic (cyclic); thio-; hydroxi; mono- or di carboxylic AA) In dependence of physical-chemical properties (acidic, basic and neutal AA) In dependence of biologic function: essential (indispensable) and nonessential (dispensable)

Aliphatical

and thio- amino acids

Aromatical, cyclic and hydroxi- AA

Neutral, acidic and basic AA

Hydrophobic nonpolar AA

Basic and acidic AA

All peptides and polypeptides are polymers of alpha-amino acids. Each of these amino acid has a different chemical structure and different properties. Each protein has a unique amino acid sequence that is genetically determined by the order of nucleotide bases in the DNA, the genetic code. Since each protein has different numbers and kinds of the twenty available amino acids, each protein has a unique chemical composition and structure.

Amino acids are joined together in proteins by peptide bonds.

Formation of peptide bond:

A peptide bond forms between the carboxyl group of one amino acid and the amino group of the adjacent amino acid.

Properties of peptide bond: The peptide bond is a rigid covalent bond and has partial doublebond character. The peptide bond is planar and unrotatable. The peptide bond is generally in the trans conformation. Each peptide bond is able to form maximum 2 hydrogen bonds with other polar atoms.

Peptide bond formation is reversible. A peptide bond can be broken by hydrolysis (the adding of water). Enzymes such as pepsin and trypsin easily hydrolyse proteins to free amino acids during digestion. However, to break peptide bonds non-enzymically requires harsh conditions (e.g. boiling in 6M HCl for 24 hours).

The products of polycondensation of a different number of -aminoacids, bound by peptide bond are named peptides: peptides: a peptide containing two amino acid units is called a dipeptide; dipeptide; one containing three amino acids, a tripeptide; tripeptide; one containing a large number, but less than 50 amino acids, a peptide; acids, peptide; one containing 50-100 amino acids 50polypeptide; if the number of aminoacids is higher then 100, 100, the polypeptide is called protein. protein.

 The end of the peptide chain with the -NH2 group is known as the N-terminal, and the end with the -COOH group is the C-terminal. A protein chain (with the N-terminal on the left) will therefore look like this:

The "R" groups come from the 20 amino acids which occur in proteins. The peptide chain is known as the backbone, and the "R" groups are known as side chains. Each protein has a specific sequence of amino acids which are assembled under the direction and control of nucleic acids.

Determination of AA in polypeptide chain

For determination of AA from N-terminal side of polypeptide chain were used: 1. - Automated Sanger Degradation 2. - Automated Edman Degradation 3. -method with dabsyl 4. - enzymatical ( aminopeptidaze) For determination of AA from C-terminal side were used: 1. Chemical method with hydrazine (methode Acabory) 2. enzymatical method (carboxipeptidaze) 3. Used the reducing agents: NaBH4 or LiBH4

. Amino Acid Sequences Can Be Determined by Automated Sanger Degradation

The peptide is hydrolyzed into its constituent amino acids by heating it in 6 N HCl at 110C for 24 hours. Amino acids in hydrolysates can be separated by ion- exchange chromatography on columns of sulfonated polystyrene. Amino acids treated with ninhydrin give an intense blue color, except for proline The concentration of an amino acid in a solution, after heating with ninhydrin, is proportional to the optical absorbance of the solution.

 The next step is often to identify the N-terminal amino acid by labeling it with a compound that forms a stable covalent bond. Fluorodinitrobenzene (FDNB) was first used for this purpose by Frederick Sanger. Dabsyl chloride is now commonly used because it forms fluorescent derivatives that can be detected with high sensitivity. It reacts with an uncharged -NH 2 group to form a sulfonamide derivative that is stable under conditions that hydrolyze peptide bonds

 Although the dabsyl method for determining the amino-terminal residue is sensitive and powerful, it cannot be used repeatedly on the same peptide, because the peptide is totally degraded in the acid-hydrolysis step and thus all sequence information is lost.

. Amino Acid Sequences Can Be Determined

by Automated Edman Degradation
The Edman degradation sequentially removes one residue at a time from the amino end of a peptide Phenyl isothiocyanate reacts with the uncharged terminal amino group of the peptide to form a phenylthiocarbamoyl derivative. The cyclic compound is a phenylthiohydantoin (PTH)-amino acid, which can be identified by chromatographic procedures. The Edman procedure can then be repeated on the shortened peptide, yielding another PTHamino acid, which can again be identified by chromatography.

Levels of Protein Structure

There are 4 levels of the protein structure:

primary secondary tertiary quaternary

The sequence of amino acids in a protein is called :

primary structure of protein

Secondary structure
is a regular arrangement of polypeptides into more compact shapes, stabilized by hydrogen bonds. The secondary structure describes the relative orientations of amino acids close in sequence. sequence. There are three predominant structure structure

alphaalpha-helix collagen-helix collagenbeta- sheets. beta- sheets.

The alpha-helix is a helical structure, a spring-like coil of

polypeptide that forms a rigid cylinder of great regularity. The alpha helix was found to be exclusively right handed. The formation of the alpha-helix is stabilized by hydrogen bonds. The hydrogen bond forms between C=O groups of one amino acid in the backbone with N-H groups located four amino acid residues further along the chain. This orientation of H-bonds produces a helical coiling of the peptide backbone such that the R-groups lie on the exterior of the helix and perpendicular to its axis. Alpha helix has 3.6 amino acids per turn of the helix. The pitch the distance separating each turn of the helix is 0,54 nm.

Collagen helix.
Another type of helix occurs in the collagens, which are important constituents of the connective tissue matrix. The collagen helix is lefthanded, and with a pitch of 0.96 nm and 3.3 residues per turn, it is steeper than the alpha-helix. The conformation is stabilized by the association of three helixes to form a right-handed collagen triple helix.

Types of secondary structure of protein

A. Alpha-helix

B.Collagen helix

Types of secondary structure of protein

Pleated-sheet structures
results from the alignment of the polypeptide backbone aside one another. Beta-sheets is stabilized by hydrogen bonds between C=O and N-H groups in different regions of the polypeptide, or even between two different polypeptides.

Identical or opposed strand alignments make up

parallel or antiparallel beta sheets.

In parallel sheets adjacent peptide chains proceed in the same direction (the direction of N-terminal to C-terminal ends is the same), whereas, in antiparallel sheets adjacent chains are aligned in opposite directions.

1. Antiparallel

2. Parallel

The side-chain groups (radicals) are not involved in alpha-helix or beta-sheet structure stabilization.

An antiparallel beta-sheet.

Tertiary protein structure

refers to the complete three-dimensional folding of the entire polypeptide chain In general, proteins fold into two broad classes of structure termed. In dependence of the proteins shape there are 2 main types of proteins: Globular proteins Fibrous proteins Globular proteins are compactly folded and coiled:

Fibrous proteins are filamentous or elongated:

Keratin fiber Collagen fiber

Stabilization of the protein's tertiary structure may involve interactions between the radicals of amino acids located far apart along the primary sequence. These may include several types of bonds:

Non-covalent Covalent - weak bonds - strong bonds

Non-covalent, weak bonds: Hydrogen bonds between side-chain groups (if the side groups contain hydroxyl or amino groups). Ionic bonds between positively and negatively charged amino acid side chains. Hydrophobic interactions (Van der Waals bonds), between the nonpolar side groups

Covalent, strong bonds:

Disulphide bridges - covalent bonds between two -SH groups of cystein to form an -S-S linkages: Cys-SH + HS-Cys Cys-S-S-Cys Estheric bonds between a side-chain carboxylic group (of asp or glu) and a sidechain hydroxyl group (of ser, tre or tir) Glu-COOH + HO-Ser Glu-CO-O-Ser Pseudo peptide bonds - between a side-chain carboxylic group (of asp or glu) and a sidechain amino group (of lys): Glu-COOH + H2N-Lys Glu-CO-HN-Lys

Bonds that stabilized tertiary structure

Hydrophobic interac

Tertiary protein structure

The tertiary structure is determined by the sequence of amino acids in the chain and is the most energetically convenient. A lot of proteins at this level begin to show their biological properties and are capable of carrying out their designated function.

Quaternary protein structure

refers to the regular association of two or more polypeptide chains to form a complex (olygomer). A multi-subunit protein may be composed of two or more identical polypeptides, or it may include different polypeptides (protomers).
Primary structure Secondary structure Tertiary structure Quaternary structure

Amino acid residues

Alpha-Helix

Polypeptide chain

Assembled subunits

Different kinds of oligomers:

Protein folding
Protein folding is the physical process by which a polypeptide folds into its characteristic and functional three-dimensional structure.

Information about the biologically active (native) conformation of proteins is already encoded in their amino acid sequences. The native forms of many proteins arise spontaneously. Nevertheless, there are special auxiliary proteins (chaperonines) that support the folding of other proteins in the conditions present within the cell

Denaturation
The native conformation of proteins can be lost as the result of denaturation: the destruction of the secondary, tertiary and quaternary structures at extreme pH values, at high temperatures, and in the presence of organic solvents, detergents, and other denaturing substances. Since denaturation reactions are not strong enough to break the peptide bonds, the primary structure (sequence of amino acids) remains the same after a denaturation process.

Effects of Denaturation

Loss of biological activity Decreased solubility Improved digestibility

Refolding or renaturation

The denatured protein can spontaneously return to its native conformation, but only if the denaturating agent was not strong enough and its action was of short duration.

Classification of proteins by chemical composition

Simple proteins - contain only amino acids

and no other chemical groups; yield only amino acids upon hydrolysis

Conjugated proteins - proteins that contain proteins

at least one prosthetic group a nonproteic structure nonproteic attached by covalent bonds or by weak interactions, required for the activity of the protein, for example the hem of protein, hemoglobin. hemoglobin. Yield, on hydrolysis, some other chemical component in addition to amino acids.

Simple proteins
Proteins that yield only alpha-amino acids by hydrolysis: albumins, globulins, histones, glutelins, prolamines, protamines.

Albumin

Globulin

Histones

Conjugated proteins proteins

proteins that contains a nonproteic structure nonproteic called its prosthetic group. Conjugated proteins are classified on the basis of the chemical nature of their prosthetic groups. Some examples of conjugated proteins are:

glycoproteins, lipoproteins, phosphoproteins, hemoproteins, flavoproteins, metalloproteins.

Glycoproteins
are generally the largest and most abundant group of conjugated proteins. They range from glycoproteins in cell surface membranes that constitute the glycocalyx, to important antibodies produced by leukocytes.

Hemoproteins

Lipoproteins Lipoproteins

Most lipids are transported in the blood as part of soluble complexes called lipoproteins
Cell membrane

Proteins differ by there physical and chemical properties:

mass Total electrical charge Termolability Solubility

Molecular

are high molecular compounds with molecular mass from 5 000 to 1 000 000 Da in dependence of the number of amino acid residues and of the number of protomers.
Proteins

depends on the presence and correlation of charged radicals of amino acids; depends of the pH of the medium

If the protein has more negatively charged amino acids (Glu and Asp) in aqueous medium its total charge will be negative. If the protein has more (Lys, Arg and His) positively charged amino acids its charge will be positive (like in histones). In acid medium the concentration of H+ is high and neutralizes the COO- - groups of amino acids - the negative charge decreases; In basic medium the concentration of OH- is high and neutralizes the positive charge of amino groups -NH3+ - the positive charge decreases.

state of the protein when its total electrical charge is equal to zero is called isoelectrical state. The value of pH when the protein is in the isoelectrical state is called isoelectrical point. The proteins in the isoelectrical state have a low solubility in water medium and can easy precipitate.
The

- the property of protein to maintain the biological activity in narrow limits of temperature (from 10 to 40C) If the temperature is higher then 50-60C the protein denatures loses its native conformation. The destruction of all the structural levels of protein (except the primary) takes place. There are several exceptions the termostable proteins (tripsin, lyzozime) stable at high temperature. If the temperature is low - the protein structure doesnt change, but the protein becomes biologically inactive.

most of proteins are hydrophilic compounds and are soluble in water. Water interacts with the polar groups of proteins and forms an aqueous membrane hydration shell - at the surface of protein
The

1. presence of the polar groups and hydration shell; 2. shape of the molecule the globular proteins are more soluble then fibrous;

3. Solvent albumins are soluble both in water and salt solution of different concentration, but globulins are not soluble in water and soluble only in weak salt solution. 4. pH of the medium pH influence on the charge of the protein; in the isoelectrical point solubility decreases.

properties Tindal effect A low speed of diffusion Osmotic (oncotic) properties A high viscosity of the solutions A capacity to form gels structural grating with water inside
Optical

precipitation Dialysis Electrophoresis Gel-filtration

Salt

The

solubility of proteins is strongly dependent on the salt concentration (ionic strength) of the medium. Proteins are usually poorly soluble in pure water. Their solubility increases as the ionic strength increases, because more and more of the well-hydrated anorganic ions are bound to the proteins surface, preventing aggregation of the molecules (salting in).

At

very high ionic strengths, the salt withdraws the hydrate water from the proteins and thus leads to aggregation and precipitation of the molecules (salting out). For this reason, adding salts such as ammonium sulfate (NH4)2SO4 makes it possible to separate proteins from a mixture according to their degree of solubility (fractionation).

Dialysis
Dialysis is used to remove lower-molecular components from protein solutions. Due to their size, protein molecules are unable to pass through the pores of a semipermeable membrane, while lower-molecular substances are able.

Electrophoresis is a technique used to separate different elements (fractions) of a blood sample into individual components. Serum protein electrophoresis is a test that measures the major blood proteins by separating them into five distinct fractions: albumin, alpha1, alpha2, beta, and gamma proteins.