Biological Chemistry Department

Biological Chemistry

Speciality: Pharmacy for foreign students (Language of instructions – English)

Lecturer: ass. prof. Kravchenko G.B.

Introduction in Biological Chemistry

Amino acids, Peptides, Proteins

Levels of Protein Structure

Physical-chemical Properties of Proteins

Lecture Plan

1. Introduction into Biochemistry. 2. Protein functions. 3. Amino acids. 3.1. Amino acid structure. 3.2. Proteinogenic amino acids classification. 4. Peptide bond and its structure. 5. Protein structure and properties. 5.1.The levels of protein structure. 5.2. Physicochemical protein properties.

Individual work

1. Structure and functions of biologically active peptides.

Information Resources

1. Biological Chemistry: Textbook / A.L. Zagayko, L.M. Voronina, G.B.

Kravchenko, K.V. Strel`chenko. – Kharkiv: NUPh; Original, 2011. – 6-21 p.

2. Training Journal for Licensed Exam “KROK-1”: Study Material in Biological

Chemistry. – Kharkiv: NUPh, 2017. – 4-11 p.

3. Laboratory Manual on Biochemistry. Kharkiv: NUPh, 2017. - 2-21 p.

4. Chemical Nature of the Amino Acids: The Medical Biochemistry Page. Available

on: https://themedicalbiochemistrypage.org/amino-acids.php.

5. Primary Structure of Proteins: The Medical Biochemistry Page. Available on:

https://themedicalbiochemistrypage.org/protein-structure.php.

Biochemistry interfaces with biology and chemistry and is concerned with the chemical processes that take place within living cells.

Modern biochemistry developed out of and largely came to replace what in the nineteenth and early twentieth centuries was called physiological chemistry, which dealt more with extracellular chemistry, such as the chemistry of digestion and of body fluids. Biochemistry as such is largely, though not exclusively, a twentieth-century discipline

Biochemistry

Statical biochemistry studies chemical structure and

biological role of mane cell compounds

Dinamical biochemistry studies metabolic ways

Proteins are a diverse and abundant class of biomolecules, constituting more than 50% of the dry weight of cells.

Proteins are the agents of biological function. Virtually every cellular activity is dependent on one or more particular proteins.

Protein functions

Functional Class Examples

Enzymes Trypsin, Pepsin, Chimitrypsin, Aldolase

Regulatory proteins Insulin, Somatotropin, Transcription

regulators

Transport protein Hemoglobin, Serum albumin,

Glucose transporter

Structural Proteins a-Keratins, Collagen

Storage proteins Ovalbumin, Casein, Ferritin

Contractile and motile proteins

Actin, Myosin

Protective and exploitive proteins

Immunoglobulin, Thrombin, Fibrinogen

Enzymes By far the largest class of proteins is enzymes. Enzymes are biological catalysts responsible for supporting almost all of the chemical reactions that maintain animal homeostasis. Virtually every step in metabolism is catalyzed by an enzyme.

Regulatory Proteins A number of proteins can regulate the ability of other proteins to carry out their physiological functions. For example, a well-known example is insulin, the hormone regulating glucose metabolism in animals. Other hormones that are also proteins include pituitary somatotropin and thyrotropin, which stimulates the thyroid gland.

Transport Proteins These proteins function to transport specific substances from one place to another. One type of transport is exemplified by the transport of oxygen from the lungs to the tissues by hemoglobin or by the transport of fatty acids from adipose tissue to various organs by the blood protein serum albumin.

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Structural Proteins Structural proteins provide strength and protection to cells and tissues. a-Keratins are insoluble fibrous proteins making up hair, horns, and fingernails. Collagen, another insoluble fibrous protein, is found in bone, connective tissue, tendons, cartilage, and hide, where it forms inelastic fibrils of great strength.

Motile Proteins Certain proteins endow cells with unique capabilities for movement. Cell division, muscle contraction, and cell motility represent some of the ways in which cells execute motion. The contractile and motile proteins underlying these motions share a common property: they are filamentous or polymerize to form filaments. Examples include actin and myosin

Protective and Exploitive Proteins Prominent among the protective proteins are the immunoglobulins or antibodies produced by the lymphocytes of vertebrates. Antibodies have the remarkable ability to “ignore” molecules that are an intrinsic part of the host organism, yet they can specifically recognize and neutralize “foreign” molecules resulting from the invasion of the organism by bacteria, viruses, or other infectious agents. Another group of protective proteins is the blood-clotting proteins, thrombin and fibrinogen, which prevent the loss of blood when the circulatory system is damaged.

Chemically, proteins are unbranched polymers of amino acids linked head to tail, from carboxyl group to amino group, through formation of covalent peptide bonds.

Central to this structure is the tetrahedral alpha (a) carbon, which is covalently linked to both the amino group and the carboxyl group. Also bonded to this a-carbon is a hydrogen and a variable side chain. It is the side chain, the so-called R group, that gives each amino acid its identity.

Amino acids structure

a-carbon

R group

Amino group Carboxyl group

There are 20 amino acids commonly found in proteins

There are several ways to classify the common amino acids. The most useful of these classifications is based on the polarity of the side chains. According this classification amino acids are grouped into the following categories: nonpolar or hydrophobic amino acids (1), neutral (uncharged) but polar amino acids (2), acidic amino acids (which have a net negative charge at pH 7.0) (3), and basic amino acids (which have a net positive charge at neutral pH) (4).

Examples

1

Alanine 2

3 4

Serine

Aspartic acid

Lysine

Peptide bond formation

The Levels of Protein Structure The architecture of protein molecules is quite complex. Nevertheless, this complexity can be resolved by defining various levels of structural organization.

Primary structure

Primary structure is fairly straightforward and refers to the number and sequence of amino acids in the protein or polypeptide chain. The covalent peptide bond is the only type of bonding involved at this level of protein structure. The sequence of amino acids in a protein is dictated by genetic information in DNA. So protein structure is genetically determined.

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Secondary structure

The secondary structure of protein molecules refers to the formation of a regular pattern of twists or kinks of the polypeptide chain. The regularity is due to hydrogen bonds forming between the atoms of the amino acid backbone of the polypeptide chain. Two main types of secondary structure have been found in proteins namely a-helix, and b-pleated sheet.

a-helix

b- pleated sheet

* Hydrogen bonds form between a hydrogen atom covalently bonded to an electronegative atom (such as oxygen or nitrogen) and a second electronegative

atom that serves as the hydrogen bond acceptor. https://lh3.googleusercontent.com/hRVxqQUPAKKRNYGSzyp4ZWE_CGq0ujV9eTZp08dEB3Nx

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a-Helix: has 3.6 amino acids per turn of the helix, which places the C=O group of amino acid №1 exactly in line with the H-N group of amino acid №5 (and C=O №2 with H-N №6).

Hydrogen bond

The peptide planes are roughly parallel with the helix axis and the dipoles within the helix are aligned, i.e. all C=O groups point in the same direction and all N-H groups point the other way. Side chains point outward from helix axis and are generally oriented towards its amino-terminal end. https://lh3.googleusercontent.com/fDkbvVBQ10s5DKGBi7ZCCfuSBeO

Cs49cWJHR_4VHN_7hNRqK41Ih_d7NIjrQmCwHlBG_3Q=s85

The b-sheet structure In a b-sheet two or more polypeptide chains run alongside each other and are linked in a regular manner by hydrogen bonds between the main chain C=O and N-H groups. Therefore all hydrogen bonds in a a-sheet are between different segments of polypeptide. This contrasts with the a-helix where all hydrogen bonds involve the same element of secondary structure. The R-groups (side chains) of neighbouring residues in a b-strand point in opposite directions.

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Tertiary structure Tertiary structure refers to the three dimensional globular structure formed by bending and twisting of the polypeptide chain. This process often means that the linear sequence of amino acids is folded into a compact globular structure.

Primary Secondary Tertiary

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The folding of the polypeptide chain is stabilized by multiple weak, noncovalent interactions. These interactions include:

Hydrogen bonds that form when a Hydrogen atom is shared by two other atoms. Electrostatic interactions that occur between charged amino acid side chains. Electrostatic interactions are attractions between positive and negative sites on macromolecules.

Hydrophobic interactions: During folding of the polypeptide chain, amino acids with a polar (water soluble) side chain are often found on the surface of the molecule while amino acids with non polar (water insoluble) side chain are buried in the interior. This means that the folded protein is soluble in water or aqueous solutions. Covalent bonds may also contribute to tertiary structure. The amino acid, cysteine, has an SH group as part of its R group and therefore, the disulfide bond (S-S ) can form with an adjacent cysteine. For example, insulin has two polypeptide chains that are joined by two disulfide bonds

The folding of the polypeptide chain is stabilized by multiple weak, noncovalent interactions. These interactions include:

Hydrogen bonds that form when a Hydrogen atom is shared by two other atoms. Electrostatic interactions that occur between charged amino acid side chains. Electrostatic interactions are attractions between positive and negative sites on macromolecules.

Hydrophobic interactions: During folding of the polypeptide chain, amino acids with a polar (water soluble) side chain are often found on the surface of the molecule while amino acids with non polar (water insoluble) side chain are buried in the interior. This means that the folded protein is soluble in water or aqueous solutions. Covalent bonds may also contribute to tertiary structure. The amino acid, cysteine, has an SH group as part of its R group and therefore, the disulfide bond (S-S ) can form with an adjacent cysteine. For example, insulin has two polypeptide chains that are joined by two disulfide bonds

1. Electrostatic interactions

2. Hydrogen bonds

3. Hydrophobic interactions

4. Disulfide bonds

5

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Hydrophobic interactions

https://i2.wp.com/rasprep.com/wp-content/uploads/2017/04/image040.jpg

http://sciencegeist.net/wp-content/uploads/2011/02/hphobichphilic.png

Quaternary structure

Quaternary structure refers to the fact that some proteins contain more than one polypeptide chain, adding an additional level of structural organization: the association of the polypeptide chains. Each polypeptide chain in the protein is called a subunit. The subunits can be the same polypeptide chain or different ones. Hemoglobin, the oxygen carrying protein in the blood, is also a tetramer but it is composed of two polypeptide chains of one type (141 amino acids) and two of a different type (146 amino acids). In chemical shorthand, this is referred to as a2ß2. For some proteins, quaternary structure is required for full activity (function) of the protein.

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eM7ADiN3ocBS7sEoXaERko7eaS7U0vt9dTbF5q8I1hOidw=s100

Four

levels

of

protein

structure

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Protein folding

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Linus Carl Pauling (1901 –1994) was an American quantum chemist and biochemist. He made important contributions to crystal and protein structure determination, and was one of the founders of molecular biology.

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Conjugated Proteins Some proteins combine with other kinds of molecules such as carbohydrates, lipids, iron and other metals, or nucleic acids, to form glycoproteins, lipoproteins, hemoproteins, metalloproteins, and nucleoproteins respectively. The presence of these other biomolecules affects the protein properties. For example, a protein that is conjugated to carbohydrate, called a glycoprotein, would be more hydrophilic in character while a protein conjugated to a lipid would be more hydrophobic in character

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Physicochemical protein properties

Size

Size of proteins is usually measured in molecular weight (mass) although occasionally the length or diameter of a protein is given in Angstroms. The molecular weight of a protein is the mass of one mole of protein, usually measured in units called daltons. One dalton is the atomic mass of one proton or neutron. The molecular weight can be estimated by a number of different methods including electrophoresis, gel filtration, and more recently by mass spectrometry. The molecular weight of proteins varies over a wide range. For example, insulin is 5,700 daltons while snail hemocyanin is 6,700,000 daltons. The average molecular weight of a protein is between 40,000 to 50,000 daltons. Molecular weights are commonly reported in kilodaltons or (kD), a unit of mass equal to 1000 daltons. Most proteins have a mass between 10 and 100 kD. A small protein consists of about 50 amino acids while larger proteins may contain 3,000 amino acids or more. One of the larger amino acid chains is myosin, found in muscles, which has 1,750 amino acids.

Charge Each protein has an amino group at one end and a carboxyl group at the other end as well as numerous amino acid side chains, some of which are charged. Therefore each protein carries a net charge. The net protein charge is strongly influenced by the pH of the solution.

Separation techniques that are based on charge include ion exchange chromatography, isoelectric focusing and chromatofocusing.

The isoelectric point (pI) is the pH at which there is no net charge on the protein. At lower pH readings, there are more positive charges in the environment and therefore, the protein has an increased cationic character. The reverse is true at pH readings above the pI.

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Hydrophobicity Literally, hydrophobic means fear of water. In aqueous solutions, proteins tend to fold so that areas of the protein with hydrophobic regions are located in internal surfaces next to each other and away from the polar water molecules of the solution. Polar groups on the amino acid are called hydrophilic (water loving) because they will form hydrogen bonds with water molecules. The number, type and distribution of nonpolar amino acid residues within the protein determines its hydrophobic character.

Hydrophilic regions are localized outside of protein molecule

Hydrophobic regions are localized inside

of protein molecule

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Solubility As the name implies, solubility is the amount of a solute that can be dissolved in a solvent. The 3-D structure of a protein affects its solubility properties. Cytoplasmic proteins have mostly hydrophilic (polar) amino acids on their surface and are therefore water soluble, with more hydrophobic groups located on the interior of the protein, sheltered from the aqueous environment. In contrast, proteins that reside in the lipid environment of the cell membrane have mostly hydrophobic amino acids (non polar) on their exterior surface and are not readily soluble in aqueous solutions. Each protein has a distinct and characteristic solubility in a defined environment and any changes to those conditions (buffer or solvent type, pH, ionic strength, temperature, etc.) can cause proteins to lose the property of solubility and precipitate out of solution. The environment can be manipulated to bring about a separation of proteins- for example, the ionic strength of the solution can be increased or decreased, which will change the solubility of some proteins.

Salting-in effect: ions shield charges and decrease protein-protein electrostatic interaction – solubility increase!

Salting-out effect: ions take “all” water – solubility decrease!

Used to selectively precipitate proteins, often with (NH4)2SO4 which is chep, effective, does not disturb structure and is very soluble.

Certain proteins precipitate from solution under conditions in which others remained quite soluble. Once protein is precipitated it can be separate.

Salting out is a method of separating proteins based on the principle that proteins are less soluble at high salt concentrations. The salt concentration needed for the protein to precipitate out of the solution differs from protein to protein. This process is also used to concentrate dilute solutions of proteins.

Salting out

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Dialysis –

-is a passage of solutes through a semi-permeable membrane. Pores in the dialysis membrane are of a certain size. Protein stays in water, salts, protein fragments, and other molecules smaller than pore size pass through.

Dialysis can be used to remove the salt if needed.

Dialysis

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The isoelectric point of a protein is the pH at which the protein has no net charge. At pH values lower than the isoelectric point, more basic side chains of amino acids become protonated to give the protein a net positive charge. At this pH solubility will be the lowest. Similarly, at pH values higher than the isoelectric point, more acidic side chains become deprotonated, giving the protein a net negative charge.

It should be noted that the protein still contains charged side chains at its isoelectric point; however, at the isoelectric point, the number of positively-charged side chains is equal to the number of negatively-charged side chains.

Isoelectric point

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Proteins can, thus, be separated according to their isoelectric point (overall charge) on a polyacrylamide gel using a technique called isoelectric focusing, which uses a pH gradient to separate proteins. Isoelectric focusing is also the first step in 2-D gel polyacrylamide gel electrophoresis.

Denaturation Proteins can denature, or unfold so that their three dimensional structure is altered but their primary structure remains intact. Many of the interactions that stabilize the protein conformation of the protein are relatively weak and are sensitive to various environmental factors including high temperature, low or high pH and high ionic strength. Protein vary greatly in the degree of their sensitivity to these factors. Sometimes proteins can be renatured but often the denaturation is irreversible.

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Electrophoresis is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field.This electrokinetic phenomenon was observed for the first time in 1807 by Reuss (Moscow State University),who noticed that the application of a constant electric field caused clay particles dispersed in water to migrate. It is ultimately caused by the presence of a charged interface between the particle surface and the surrounding fluid.

Electrophoresis

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Conclusions

1. Chemically, proteins are unbranched polymers of a-amino acids linked from carboxyl group to amino group, through formation of covalent peptide bonds. 2. Biological functions of proteins: enzymes, transport proteins, nutrient and storage proteins, contractile or mobile proteins, structural proteins, defence proteins, regulatory proteins, etc. 3. There are 20 a-amino acid that are relevant to the make-up of mammalian proteins. 4. There are several ways to classify the common amino acids: structural, physico-chemical and biological. 5. Every protein has unique three-dimensional structure that reflects its function . 6. There are two general classes of proteins: fibrous and globular.

Do you have any questions?

Thank you for your attention!

https://www.youtube.com/watch?v=JxK5rZxbyQY