Lecture -1

CHEMISTRY OF PROTEINS By/Assistant professor Dr. Naglaa F. Khedr

1 Objectives

Functions of proteins

Amino acids, types, properties

Classification of amino acids

Protein structures

2 Biological Functions of Proteins

1. Enzymes: regulate metabolism by selectively accelerating chemical reactions ( e.g. Ribonuclease) 2. Hormones – e.g. Insulin 3. Transport proteins – e.g. Hemoglobin, albumin. 4. Structural and support proteins – e.g. Collagen. 5. Contractile proteins – e.g. Actin & Myosin. 6. Immunity : defense against foreign substances ( e.g. Immunoglobulins) 7. Exotic proteins – e.g. Antifreeze proteins in fish. 3 Amino Acids are Building Blocks of Proteins R Different side chains, R, determine the properties of 20 amino acids. + NH3 C COO-

Amino group Carboxylic H acid group

Amino acids are the monomeric units or "building blocks" of proteins that are joined together covalently in peptide bonds, i.e., proteins are polymers of amino acids.

.Amino acids are composed of amine (NH2) and carboxylic acid (COOH) functional groups, along with a side-chain specific to each amino acid. • Only 20 are commonly found as constituents of mammalian proteins. [Note: These are the only amino acids that are coded for by DNA, the genetic material in the cell • All amino acids have a common structural unit that is built around the alpha carbon. L-α-amino acids

COOH  H2N C H R R • R groups are the only variable groups in the structure

All α-amino acids, except glycine, have an asymmetric carbon and thus, they have two enantiomers; D and L.

 L form (protonated amine group can be on the left-hand)  D form (protonated amine group can be on the right-hand) 5 Amino Acid Classification

6 Aliphatic Side-Chain Amino Acids

7 Hydroxy-Containing Amino Acids

Sulfur-Containing Amino Acids Basic Amino Acids

Acidic Amino Acids and their Amides

9 Amino Acids with aromatic ring

Heterocyclic Amino Acids

10 Amino Acids Not Found in Proteins

S-Adenosylhomocysteine Citrulline L-Homoserine

S-Adenosylmethionine β-Cyanoalanine L-Ornithine

β-Alanine D-Glutamic Acid Sarcosine -Amino butyric acid D-Alanine L-Thyroxine (GABA) Azaserine Homocysteine

11 Posttranslational modification of amino acids

. Some amino acids are chemically modified after they are incorporated into proteins. . The results of these modifications produce the following residues found in proteins: . O-Phosphoserine (in active sites of enzymes) . 4-Hydroxyproline (collagen) . -Hydroxylysine (collagen) . Thyroxine (hormone) . -Carboxyglutamic acid (blood clotting)

12 Classification of amino acids into; hydrophobic and hydrophilic amino acids

 The hydrophobic amino acids; tend to repel the aqueous environment and, therefore, reside predominantly in the interior of proteins. This class of amino acids does not ionize nor participate in the formation of H-bonds.  The hydrophilic amino acids; tend to interact with the aqueous environment, are often involved in the formation of H-bonds and are predominantly found on the exterior surfaces proteins or in the reactive centers of enzymes. 13 Amino acids

Hydrophilic (polar) Hydrophobic (non-polar)

Aliphatic side chain Aromatic side negatively charged positively charged uncharged e.g. chain e.g. e.g. glycine e.g. serine e.g. aspartic acid lysine alanine threonine Tyrosine glutamic acid arginine valine aspargine leucine histidine glutamine phenyl alanine isoleucine tryptophan cysteine proline methionine Hydrophilic amino acids re found in On the outside of proteins that function in an aqueous environment and in the interior of membrane-associated Hydrophobic amino acids re found in proteins the interior of proteins that function in an aqueous environment and on the surface of proteins (such as membrane proteins) that interact with lipid

14 Classification of amino acids according to the synthesis in the body

 Dietary amino acids are classified as: essential (must be in the diet and cannot be synthesized by the organism). or non-essential (can be synthesized by the organism).

Essential Amino Acids in Non-Essential Amino Acids in Mammals Mammals Arginine, Histidine, Isoleucine, Alanine, Asparagine, Aspartic Leucine, Lysine, Methionine, Acid, Cysteine, Glutamic Acid, Phenylalanine, Threonine, Glutamine, Glycine, Proline, Tryptophan, Valine Serine, Tyrosine

Arginine and Histidine are semi-essential amino acids.

15 Acid –base properties and Ionization of amino acids  Due to ionizing property of amino acids, amino acids exert acid- base behavior and Amphoteric properties (zwitter ion formation).  At physiological pH (around 7.4), the carboxyl group will be deprotonated and the amino group will be protonated. An amino acid with no ionizable R-group would be electrically neutral at this pH.  This species is termed a zwitter ion (substance containing equal numbers of positive and negative charges).  The net charge will depend upon the pH of the surrounding aqueous environment  the pH at which zwitter ion is formed will be the isoelectric point: pI.

16 The α-COOH and α-NH2 groups in amino acids are capable of ionizing (as are the acidic and basic R-groups of the amino acids). R-COOH <------> R-COO- + H+ + + R-NH3 <------> R-NH2 + H

H

+ - NH3 C COO R What is zwitter ion? Zwitter ion Isoelectric pH Net charge zero At pH=7.4

17

Isoelectric point

• The isoelectric point (pI), is defined as pH at which a particular molecule carries no net electrical charge.

18 Name the structure of the following amino acids

19 Erythrocytes rhinoceros fireflies

20 Peptides and Proteins . 20 amino acids are commonly found in protein. . Peptide bond formation is a condensation reaction leading to the polymerization of amino acids into peptides and proteins. . Peptides and proteins (what’s the difference?). - The chains containing less than 50 AA are called “peptides”, while those containing greater than 50 AA are called “proteins”.

21 Peptide bond formation: .α-carboxyl group of one amino acid forms a covalent peptide bond with α-amino group of another amino acid by removal of a molecule of water. .Repetition of this process generates a polypeptide or protein of specific amino acid sequence.

22 Peptides are chains of amino acids

Pentapeptide

hydrolysis condensation

Serylglyciltyrosylalanylleucine or Two amino acid molecules can be Ser-Gly-Tyr-Ala-Leu covalently joined through a or substituted amide linkage, termed a SGYAL peptide bond, to yield a dipeptide

The free amino end (N-terminal) of the peptide is written to the left and the free carboxyl (C-terminal) to the right. Therefore all amino acids are read from N to C terminal Dipeptide contains 2 amino acids, Oligopeptide contains 12-20 amino acids, Polypeptide contains many amino acids, and Proteins are polypeptides of greatly divergent length. 23 In the peptide bond:  the -C=O and the -N-H bonds are nearly parallel and the C, O, N, and H atoms are usually coplanar.  There is little twisting (rotation) possible around the C-N bond because the peptide bond has a partial double bond character. Thus, peptide bond is rigid and planar.  The atoms about the peptide bond usually exist in the trans configuration to minimize the steric interaction between bulky R groups on adjacent α- carbon atoms.

Partial double bond character Cis & Trans Configuration

24 Levels of Protein Structures

Classified into four categories 1. Primary structure 2. Secondary structure 3. Tertiary structure 4. Quaternary structure

1- Primary structure • The sequence of amino acids in a protein is called the primary structure of the protein and refers to the linear number and order of the amino acids present. • The two ends of the polypeptide chain are referred to as the carboxyl terminus (C-terminus) and the amino terminus (N-terminus). • Counting of residues always starts at the N-terminal

end (NH2-group). • The primary structure of a protein is determined by inherited genetic information. • The sequence of a protein is unique to that protein, the primary structure determines the chemical characteristics and conformation of protein, which determines the function.

2- Secondary structure The secondary structure describes regular ways to fold the polypeptide chain, e.g. α-Helix and β-Pleated sheets.

27 I. Alpha-Helix: • It is a spiral structure, consisting of a tightly packed, coiled polypeptide backbone core, with the side chains of amino acids extending outward from the central axis to avoid steric interference with each other. • The formation of the α-helix is spontaneous and is stabilized by H bonds between peptide bonds spaced four-residues apart. Each turn of α-helix contains 3.6 amino acids. • ex. Keratins mainly found in hair and skin ii) -pleated sheet structure or stretched state structure

. In the β-sheet, all of the peptide bond components are involved in hydrogen bonding. The folding and alignment of stretches of the polypeptide backbone aside one another to form β-sheets stabilized by H-bonding.

 A β-sheet can be formed from two or more separate polypeptide chains or segments of polypeptide chains.  Beta sheets are said to be pleated. This is due to positioning of the α-carbons of the peptide bond, which alternates above and below the plane of the sheet.  Beta sheets are either parallel or antiparallel. In parallel sheets, adjacent peptide chains proceed in the same direction, whereas, in antiparallel sheets, adjacent chains are aligned in opposite directions.

30 • The structural properties of silk are due to beta pleated sheets. – The presence of so many hydrogen bonds makes each silk fiber stronger than steel.

31 3) Tertiary structure

 It results from the folding of a polypeptide into closely packed three dimensional structure. • The tertiary structure is determined by a variety of bonding interactions between the "side chains" on the amino acids. • These bonding interactions may be stronger than the hydrogen bonds between amide groups holding the helical structure. • As a result, bonding interactions between "side chains" may cause a number of folds, bends, and loops in the protein chain. • There are four types of bonding interactions between "side chains" including: hydrogen bonding, salt bridges, disulfide bonds, and non-polar hydrophobic interactions.

4-Quaternary structure of protein  The proteins that have more than one polypeptide chain (two or more subunits, oligomeric) in their native conformation have a quaternary structure.  Many proteins consist of a single polypeptide chain and are called monomeric proteins.  Many other consist of two or more polypeptide chains called oligomeric proteins.  The arrangement of these polypeptide in three dimensional complexes is called the quaternary structure of the protein. they are held together by non-covalent interactions or forces such as:- 1- Hydrophobic interactions. 2- Hydrogen bond 3- lonic bonds. •Oligomeric proteins can be composed of multiple identical polypeptide chains (homo-oligomers) or multiple distinct polypeptide chains (hetero-oligomers). •Example: Hemoglobin is a hetero-oligomeric protein because it contains two α and two β subunits arranged with a quaternary structure in the form α2β2.

Quaternary Structure of Hemoglobin 34 Bonds Holding Protein Structure: i) Disulfide Bond: The formation of disulfide bonds between cysteines present within proteins produces Cystine.

35 ii) Hydrogen Bonding: H-bonding occurs between proton donors and acceptors, not only within and between polypeptide chains, but also with the surrounding aqueous medium. iii) Electrostatic or ionic bond or salt bond or salt bridge:- These are formed between oppositely charged groups when they are close. Such + as amino (NH3 ) terminal and carboxyl (COO-) terminal groups of the peptide and the oppositely charged R-groups of polar amino acid residues.

36 iv) Hydrophobic Forces: The hydrophobicity of certain amino acid R-groups tends to drive them away from the exterior of proteins and into the interior. v) Van der Waal`s Forces: There are both attractive and repulsive van der Waal`s forces that control protein folding. The repulsion is the result of the electron-electron repulsion that occurs as two clouds of electrons begin to overlap.

37 Denaturation of Proteins  Protein denaturation results in the unfolding and disorganization of the protein's secondary and tertiary structures, which are not accompanied by hydrolysis of peptide bonds, i.e. the primary structure remains intact.  Denaturation destroys weak bonds and disulfide bonds.  Denaturation may, under ideal conditions, be reversible, in which case the protein refolds into its original native structure when the denaturing agent is removed. However, in most cases, it is irreversible.  Denatured proteins are often insoluble and, therefore, precipitate from solution.

38 Question

why boiling makes an egg white turn opaque?

39 (1) Agents that disrupt hydrogen bonding: Heat (thermal agitation or vibration). Urea and guanidinium chloride: At high concentration (8 to 10 M for urea, and 6 to 8 M for guanidinium chloride), they compete for the hydrogen bonds of the native structure.

(2) Agents that disrupt hydrophobic interaction: Organic solvents, such as acetone or ethanol, dissolve nonpolar groups. Detergents, such as SDS, also dissolve nonpolar groups.

(3) Agents that disrupt electrostatic interaction: Extremes of pH result in large net charges, resulting in intramolecular electrostatic repulsion, which will favor an extended conformation rather than a compact one.

40 (4) Agents that disrupt disulfide bridges: i) Oxidation of a disulfide by performic acid results in the formation of two equivalents of cysteic acid. Electrostatic repulsion between

- SO3 groups prevents S-S recombination.

41 ii) Reduction of a disulfide by either 2-mercaptoethanol or dithiothreitol yields –SH groups. Iodoacetic acid is added to alkylate the free sulfhydryls to prevent reformation of the disulfide bonds.

42 43