proteins lecture notes.

Proteins and Amino Acids Dr. Sebnem GARIP USTAOGLU  PROTEINS The central Dogma of Biology DNA RNA Protein DNA: AAA GGA GAA TAG GGC RNA: UUU CCU CUU AUC CCG Protein: Phe Pro Leu Ile Pro (FPLIP) Biomedical Importance of Proteins Enzymes...

Proteins and Amino Acids Dr. Sebnem GARIP USTAOGLU  PROTEINS The central Dogma of Biology DNA RNA Protein DNA: AAA GGA GAA TAG GGC RNA: UUU CCU CUU AUC CCG Protein: Phe Pro Leu Ile Pro (FPLIP) Biomedical Importance of Proteins Enzymes Immunoglobulins Hormones Mechanical support Receptors Transport; albumin Storage proteins; ferritin Oxygen binding proteins; hemoglobin, myoglobin Energy source (not mainly) Oncotic pressure  Composition of Proteins In addition to C, H, and O which are present in carbohydrates and lipids, proteins also contain N. Small amounts of S and P are also present. Few proteins contain other elements such as I, Cu, Mn, Zn and Fe, etc. The monomers of proteins are called as amino acids. Proteins are made up from, 20 standard amino acids in different sequences and numbers.  Amino Acids Proteins are the unbranched polymers of L- α- amino acids. Proteins contain only L-α-amino acids. In most of the amino acids, an amino group is attached to α-carbon atom next to the carboxyl group hence they are α-amino acids. Amino Acids  Amino Acids Only 20 amino acids serve as building blocks of body proteins. They are known as common amino acids. Some proteins contain additional amino acids that arise by modification of an amino acid already present in a peptide (post translational modification); 4 hydroxyproline and 5-hydroxylysine; -carboxyglutamate  Properties of Amino Acids Size and Shape Charge Polarity Hydrophobicity Aromaticity Conformation-usually determined by side chain  Classification and Structure of Amino Acids Neutral, Acidic & Basic amino acids Hydrophobic & Hydrophilic amino acids Polar & Nonpolar amino acids Aliphatic & Aromatic amino acids Sulphur containing amino acids Imino acid-Proline  Two special amino acid Smallest amino acid; (Aliphatic, non-polar, hydrophobic) Glycine Imino acid; (Aliphatic, non-polar, hydrophobic) Proline  Aliphatic Amino Acids C, H containing amino acids in their side chains (do not contain hetero atoms; N, O or S) Glycine, Alanine, Valine, Leucine, Isoleucine, Proline (also imino acid) Aliphatic Amino Acids Hydrophobic (Non-polar) Amino Acids Mostly do not contain O, N groups in their side chains In addition to aliphatic amino acids, Methionine (also sulphur containing amino acid) Phenylalanine, tyrosine (also aromatic amino acids) Tryptophan (also aromatic and heterocyclic amino acid)  Acidic Amino Acids Carboxyl group containing amino acids in their side chains Completely ionized and negatively charged at physiologic pH Aspartic acid, glutamic acid Acidic Amino Acids  Basic Amino Acids Amino group containing amino acids in their side chains Completely ionized and positively charged at physiologic pH Lysine, arginine Histidine in deprotonated form (exception- also aromatic amino acid in its protonated form in physiologic pH-acidic) Basic Amino Acids  Aromatic Amino Acids Phenylalanine, tyrosine Tryptophan (also heterocyclic amino acid) In physiologic pH, histidine is also aromatic Absorb UV light at 280 nm  Find the net charge of Methionine in pH=11. pKa value for COOH= 2,3 pKa value for NH3= 9,2  Nutritional Importance Essential and non-essential amino acids Essential amino acids; preferably not synthesized in the body, obtained from the diet. Methionine (M), arginine (A), tryptophan (T), threonine (T), valine (V), isoleucine (IL), leucine (L), phenyl alanine (P), histidine (H) and Lysine (L). Sometimes histidine and arginine are referred as semi-essential because body synthesizes these amino acids to some extent.  Nutritional Importance Non-essential amino acids; are synthesized in the body. Alanine, glycine, serine, tyrosine, glutamate, glutamine, aspartate, aspargine, cysteine and proline. Biosynthesis of Nonessential Amino Acids Nonessential AAs Essential AAs Alanine Arginine Asparagine Histidine Aspartate Isoleucine Cysteine Leucine Glutamate Lysine Glutamine Methionine Glycine Phenylalanine Proline Threonine Serine Tryptophan Tyrosine Valine Essential Amino Acids are Difficult to Synthesize Number of the enzymes required: Essential AAs Non-essential AAs Arg 7 Ala 1 His 9 Asp 1 Ile 8 Asn 1 Leu 7 Cys 2 Lys 8 Glu 1 Met 5 Gln 1 Phe 10 Gly 1 Thr 6 Pro 3 Trp 13 Ser 3 (1) Val 8 Tyr 1 Tyr 10  Rare & Unusual Amino acids Not found in proteins but play important roles in metabolism. 1. Ornithine, citrulline and arginino succinic acid; urea cycle. 2. β-alanine ; co-enzyme A. 3. Taurine; bile acids. 4. γ-aminobutyric acid; neurotransmitter. 5. Mono- and di-iodotyrosine; precursors of thyroxine. 6. Pantothenic acid; water-soluble vitamin (vitamin B5). 7. Homoserine; methionine catabolism. 8. Homocysteine; methionine catabolism. It triggers platelet adhesion. Hence, it is considered as a risk factor for development of coronary artery disease (CAD). 9. S-allylcysteine sulfoxide ; obtained from garlic. It has many therapeutic effects. It is commonly called as alliin.  New Amino Acids Recently two more new amino acids described for protein synthesis. Selenocysteine - 21st amino acid. occurs at the “active site” of several enzymes. Pyrrolysine - 22nd amino acid. The STOP codon UAG can code for pyrrolysine. Pyrrolysine is not found in human, found in archae and bacteria.  Peptide Linkage The amino acid components of peptides and proteins are linked together by amide bonds (peptide bonds) between α-carboxyl and α- amino groups. Amino terminal Carboxy terminal (N terminus) (C terminus)  Peptide Linkage Chains that contain fewer than 50 amino acid residues are called peptides or oligopeptides. To express the sequence of a peptide; the three-letter or single-letter abbreviations for the amino acid residues. Sequence always starts at the N terminus. The number and order of all of the amino acid residues in a polypeptide constitute its primary structure.  Biomedically Important Peptides Aspartame; sweetening agent. Glutathione; antioxidant system. Chemotactic peptide; It plays an important role in chemotaxis in leukocytes. Enkaphalin; It binds to opiate receptors present in brain. Angiotensin II; a powerful vasoconstrictor and raises blood pressure. Bradykinin;. a powerful vasodilator and anti inflammatory. Oxytocin; stimulates uterus contraction. Vasopressin; It is also known as antidiuretic hormone (ADH). Increases water re-absorption in the kidney and vasoconstrictor. Structural Organization of Proteins Protein structure is normally described at four levels of organisation; primary, secondary, tertiary and quaternary. Primary structure; the sequence of the amino acids in a polypeptide chain.  Secondary Structure The folding of short (3- to 30-residue), segments of polypeptide into geometrically ordered units. The linkages or bonds involved in the secondary structure formation are; Hydrogen bonds: weak, low energy noncovalent bonds. Hydrogen bonds are formed in secondary structure by sharing H-atoms between oxygen of CO and nitrogen of -NH of different peptide bonds. The hydrogen bonds in secondary structure may form either an α-helix or β-pleated sheet structure. Alpha Helix Structure intra chain hydrogen bonds right handed α-helix  Beta Sheet Structure When the adjacent polypeptide chains run in same direction (N to C terminus) the structure is termed as parallel β-pleated sheet. When the adjacent polypeptide chains run in opposite direction the structure is termed as anti-parallel β-pleated sheet. parallel β-pleated sheet anti-parallel β-pleated sheet  Random Coil (Disordered) Conformation Regions of proteins that are not organized as helices and pleated sheet are said to be present in random coil conformation. These are also equally important for biological function of proteins as those of helices and β- pleated sheet.  β-turn or β-bends (Reverse Turn) Hair pin turn of a polypeptide chain is called as β-turn. The change in the direction of a polypeptide chain is achieved by β-turn. β-turn connects anti parallel β-sheets. Usually four aminoacids make up β-turn; Gly, Ser, Asp, proline.  Super Secondary Structure In some globular proteins regions of α-helix and β-pleated sheet join to form super secondary structure or motifs. Motif  Tertiary Structure three-dimensional folding of polypeptide chain Tertiary structure of a protein is mainly stabilized by non-covalent bonds  Non-covalent Bonds in Tertiary Structure Some type of non-covalent bonds which stabilize protein structure (a) electrostatic interaction, (b) hydrogen bonding between tyrosine residues carboxylate groups on side- chains, (c) interactions of non-polar side chains caused by the mutual repelsion of the solvent, (d) van der Waals interactions.  Disulfide bridges (in some proteins): disulfide bond formed between two cysteine residues. They are strong, high energy covalent bonds.  Quaternary Structure Proteins containing two or more polypeptide chains possess quaternary structure. These proteins are called as oligomers. The individual polypeptide chains are called as protomer, monomers or subunits. The protomers are united by electrostatic interactions other than covalent bonds. Occasionally, they may be joined by disulfide bonds.  Quaternary Structure Haemoglobin consist of 4 polypeptide chains. Hexokinase contains 2 subunits. Pryuvate dehydrogenase contains 72 subunits.  Forces in Quaternary Structure 1. Hydrogen bonding 2. Electrostatic interactions 3. Hydrophobic interactions 4. Vander waals interactions 5. Disulfide bonds  Primary Structure: sequence of residues Secondary Structure: localized folding Tertiary Structure: complete folding pattern Quaternary Structure: interaction of subunits  Protein Folding Protein Folding Enzymes (a) Disulfide isomerase; In the newly formed protein molecules –SH groups of cysteine residues may form several intra or inter disulfide linkages. However, only few disulfide linkages may be essential for proper protein folding. The disulfide isomerase favours formation of such disulfide linkages by breaking unwanted linkages formed. (b) Cis-trans prolyl isomerase; It aids folding process by catalyzing inter conversion of cis-trans peptide bonds of proline residues of folding protein.  Protein Folding Protein Factors Chaperons (Chaperonins) These proteins aid protein folding process by preventing formation of aggregates. Chaperons accelerate protein folding by blocking protein folding pathways of unproductive nature. They bind to hydrophobic parts of protein molecules and prevent formation of aggregates.  Denaturation of Proteins Denaturation is loss of native conformation. On denaturation, physical, chemical and biological properties of a protein are altered. Denatured protein=unfolded protein=inactive protein  Causes of Denaturation 1. High temperature 2. Extreme alkaline or acidic pH 3. Use of urea and guanidine at high concentration 4. UV radiation 5. Sonication 6. Vigorous shaking 7. Detergent like sodium dodecylsulfate also denatures protein 8. Treatment with organic solvents like ethanol, acetone etc. 9. Treatment with strong acids like trichloro acetic acid, picric acid and tungstic acid 10. Exposure to heavy metals like Pb2+, Ag2+ and Cu2+ CLASSIFICATION OF PROTEINS ON THE BASIS OF SHAPE AND SIZE Fibrous proteins: When the axial ratio of length: width of a protein molecule is more than 10, it is called a fibrous protein. α-keratin from hair and collagen are in this group. Globular protein: When the axial ratio of length: width of a protein molecule is less than 10, it is called as globular protein. myoglobin, hemoglobin  Structural Protein: Collagen It is the major protein comprising the ECM. In mammals, 25% of the total protein is collagen. Collagens are mainly synthesized by fibroblasts (cells found in connective tissue), muscle cells and epithelial cells. There are about 28 different types of collagen. Type I, II and III are the most abundant types.  Types of Collagen Each collagen is a triple helix of one or two different polypeptide chains. Structures include fibrils and networks. Collagenases degrade the collagen.  Collagen Structure It consists of 3 coiled subunits (3 polypeptide chains-α chains). Each chain consists of about 1000 amino acids twisted around each other in a characteristic right-handed Triple helix. There are 3 amino acids per turn of the helix. Every third amino acid residue of collagen is glycine (Gly). Thus collagen is a polymer of (G-X-Y) repeats, where X and Y may be proline (10%) and 4- hydroxyproline (10%) or 5-hydroxylysine (1%), respectively. Collagen type I contains approx. 33% Gly and 21% proline and hydroxyproline.  Once three of these polypeptides have become ‘braided’ to form a triple helix, hydrophobic interactions (between glycines) and hydrogen bonds (between 4- hydroxyproline) stabilize the conformation. Intra and inter-chain Covalent bonds formed by 5-hydroxylysine residues, enable strength and hardness of the molecules. Molecular features of collagen structure from primary sequence up to the fibril covalent cross links formed between lysine and hydroxylysine residues THANK YOU