Protein Chemistry- 1 PDF

Protein Chemistry-1 Samreen Sheik, Department of Biochemistry Manipal University College Malaysia (MUCM) Learning Outcomes Proteins (SLS) o Define the term peptide bond o Explain the properties of peptide bond o Mention the significance of few physiologically important peptides o Show...

Protein Chemistry-1 Samreen Sheik, Department of Biochemistry Manipal University College Malaysia (MUCM) Learning Outcomes Proteins (SLS) o Define the term peptide bond o Explain the properties of peptide bond o Mention the significance of few physiologically important peptides o Show the 'N' and 'C' terminals of a peptide o Illustrate with an example the nomenclature of peptides Classify proteins based on composition, functions and shape with examples List the differences between globular and fibrous proteins List the protein purification techniques with the basis of separation https://www.youtube.com/watch?v=gG7uCskUOrA&t=11s Is protein important? Protein are the most abundant organic molecules of the living system. They occur in every part of the cell and constitute about 50% of the cellular dry weight. Proteins form the fundamental basis of the structure and function of life. Abnormality in protein structure will lead to molecular diseases with profound alterations in metabolic functions. Proteins are the polymers of amino acids linked together by a peptide bond Peptide Bond Peptide bonds are anhydride( i.e formed by the loss of a water molecule), covalent bonds formed between a carboxyl group of an amino acid and an amino group of succeeding amino acid. Properties of Peptide Bond Are responsible for the polymerization of amino acids to form peptides ‘N’ and ‘C’ terminal of a peptide 1. In a polypeptide chain, at one end there will be one free alpha amino group. This end is called the amino terminal (N-terminal) end and the amino acid contributing the alpha-amino group is named as the first amino acid. 2. Usually the N-terminal amino acid is written on the left hand side when the sequence of the protein is denoted. Incidentally, the biosynthesis of the protein also starts from the amino terminal end. 3. The other end of the polypeptide chain is the carboxy terminal end (C-terminal), where there is a free alpha carboxyl group which is contributed by the last amino acid. All other alpha amino and alpha carboxyl groups are involved in peptide bond formation.  Nomenclature of peptides The free amino end (N-terminal) of the peptide chain is written to the left and the free carboxyl end (C-terminal) to the right. Therefore, all amino acid sequences are read from the N- to the C-terminal end. In Fig. A, the order of amino acids in the dipeptide is valine, alanine. In proteins, the carboxyl group of one amino acid links with the amino group of another amino acid to form a peptide. This results in the removal of water and what remains is called the residue. When a peptide is named, all amino acid residues have their suffixes (-ine, - an, - ic, or -ate) changed to -yl, with the exception of the free C-terminal amino acid. For example, a tripeptide composed of an N-terminal valine, a glycine, and a C- terminal leucine is called valylglycylleucine. Significance of few physiologically important peptides 1. Glutathione : It is a tripeptide composed of 3 amino acids. Chemically, glutathione is gamma glutamyl-cysteinyl-glycine. Functions Glutathione (reduced) performs specialized functions in erythrocytes (i) It maintains RBC membrane structure and integrity. (ii) It protects hemoglobin from getting oxidized by agents such as H2O2. Glutathione is involved in the transport of amino acids in the intestine and kidney tubules via gamma-glutamyl cycle or Meister cycle Glutathione is involved in the detoxication process. Toxic amounts of peroxides and free radicals produced in the cells are scavanged by glutathione peroxidase (a selenium containing enzyme). Glutathione serves as a coenzyme for certain enzymes e.g. prostaglandin PGE2 synthetase, glyoxylase. 2. Thyrotropin releasing hormone (TRH) : It is a tripeptide secreted by hypothalamus. TRH stimulates pituitary gland to release thyrotropic hormone. 3. Oxytocin : It is a hormone secreted by posterior pituitary gland and contains 9 amino acids (nonapeptide). Oxytocin causes contraction of uterus. 4. Vasopressin (antidiuretic hormone, ADH) : ADH is also a nonapeptide produced by posterior pituitary gland. It stimulates kidneys to retain water and thus increases the blood pressure. 5. Angiotensins : Angiotensin I is a decapeptide (10 amino acids) which is converted to angiotensin II (8 amino acids). The later has more hypertensive effect. Angiotensin II also stimulates the release of aldosterone from adrenal gland. 6. Methionine enkephalin : It is a pentapeptide found in the brain and has opiate like function. It inhibits the sense of a pain. 7. Bradykinin and kallidin : They are nona and decapeptides, respectively. Both of them act as powerful vasodilators. They are produced from plasma proteins by snake venom enzymes. 8. Peptide antibiotics : Antibiotics such as gramicidin, bacitracin, tyrocidin and actinomycin are peptide in nature. 9. Aspartame : It is a dipeptide (aspartylphenylalanine methyl ester), produced by a combination of aspartic acid and phenylalanine. Aspartame is about 200 times sweeter than sucrose, and is used as a low-calorie artificial sweetner in softdrink industry. 10. Gastrointestinal hormones : Gastrin, secretin etc. are the gastrointestinal peptides which serve as hormones. Classification of protein based on the amino acid composition, structure, shape and solubility properties. 1. Simple proteins : They are composed of only amino acid residues. 2. Conjugated proteins : Besides the amino acids, these proteins contain a non- protein moiety known as prosthetic group orconjugating group. 3. Derived proteins : These are the denatured or degraded products of simple and conjugated proteins. (a) Globular proteins : These are spherical or oval in shape, soluble in water or other solvents and digestible. (i) Albumins : Soluble in water and dilute salt solutions and coagulated by heat. e.g. serum albumin, ovalbumin (egg), lactalbumin (milk). (ii) Globulins : Soluble in neutral and dilute salt solutions e.g. serum globulins, vitelline (egg yolk). (iii) Glutelins : Soluble in dilute acids and alkalies and mostly found in plants e.g. glutelin (wheat), oryzenin (rice). (iv) Prolamines : Soluble in 70% alcohol e.g. gliadin (wheat), zein (maize). (v) Histones : Strongly basic proteins, soluble in water and dilute acids but insoluble in dilute ammonium hydroxide e.g. thymus histones. (vi) Globins : These are generally considered along with histones. However,globins are not basic proteins and are not precipitated by NH4OH. (vii) Protamines : They are strongly basic and resemble histones but Smaller in size and soluble in NH4OH.They are also found in association with nucleic acids e.g. sperm proteins. viii.Lectins: are carbohydrate-binding proteins, and are involved in the interaction between cells and proteins.They help to maintain tissue and organ structures. e.g. agglutinin. (b)Fibrous proteins/ scleroproteins : These are fiber like in shape, insoluble in water and resistant to digestion. (i) Collagens are connective tissue proteins lacking tryptophan. (ii) Elastins : These proteins are found in elastic tissues such as tendons and arteries. (iii) Keratins : These are present in exoskeletal structures e.g. hair, nails,horns. Human hair keratin contains as much as 14% cysteine 2. Conjugated proteins (a) Nucleoproteins : Nucleic acid (DNA or RNA) is the prosthetic group e.g. nucleohistones,nucleoprotamines. (b) Glycoproteins : The prosthetic group is carbohydrate, which is less than 4% of protein.e.g. mucin (saliva), ovomucoid (egg white). (c) Lipoproteins : Protein found in combination with lipids as the prosthetic group e.g. serum lipoproteins. (d) Phosphoproteins : Phosphoric acid is the prosthetic group e.g. casein (milk), vitelline (egg yolk). (e) Chromoproteins : The prosthetic group is coloured in nature e.g. hemoglobins, cytochromes. (f) Metalloproteins : These proteins contain metalions e.g. ceruloplasmin (Cu), carbonic anhydrase (Zn). 3. Derived proteins: 1.The primary derived are the denatured or coagulated or first hydrolyzed products of proteins. (i) Coagulated proteins : These are the denatured proteins produced by agents such as heat, acids, alkalies etc. e.g. cooked proteins, coagulated albumin (egg white). (ii) Proteans : These are the earliest products of protein hydrolysis by enzymes, dilute acids, alkalies etc. which are insoluble in water. e.g. fibrin formed from fibrinogen. (iii) Metaproteins : These are the second stage products of protein hydrolysis obtained by treatment with slightly stronger acids and alkalies e.g. acid and alkali metaproteins. 2.The secondary derived are the degraded (due to breakdown of peptide bonds) products of proteins. These include proteoses, peptones, polypeptides and peptides.  Nutritional classification of proteins 1. Complete proteins : These proteins have all the ten essential amino acids in the required proportion by the human body to promote good growth. e.g. egg albumin, milk casein. 2. Partially incomplete proteins : These proteins partially lack one or more essential amino acids, and can promote moderate growth. e.g. wheat and rice proteins (limiting Lys, Thr). 3. Incomplete proteins : These proteins completely lack one or more essential amino acids. Hence they do not promote growth at all e.g. gelatin (lacks Trp). Classification of protein based on Function 1. Structural proteins : Keratin of hair and nails, collagen of bone. 2. Enzymes or catalytic proteins : Hexokinase, pepsin. 3. Transport proteins : Hemoglobin, serum albumin. 4. Hormonal proteins : Insulin, growth hormone. 5. Contractile proteins : Actin, myosin. 6. Storage proteins : Ovalbumin, glutelin. 7.Genetic proteins : Nucleoproteins. 8. Defense proteins : Snake venoms, Immunoglobulins. 9. Receptor proteins for hormones, viruses. Differences between globular and fibrous proteins List the protein purification techniques with the basis of separation. 1. Purifying protein from extraction buffer 2. Electrophoretic techniques 3. Other techniques  Purifying protein from extraction buffer Name of the technique Property of the protein Ion Exchange Chromatography Overall Charge Gel filtration Chromatography Molecular size Affinity chromatography Specific ligand binding properties Adsorption chromatography Adsorption property Electrophoretic techniques Name of the technique Property of the protein Polyacrylamide Gel Molecular Size Electrophoresis (PAGE) Isoelectric focusing Charge (the protein stops Movement in the gel at its isoelectric pH) 2-Dimentional Molecular size and charge electrophoresis Gradient Gel Molecular size Electrophoresis Other techniques Property of the protein Name of the technique Ultracentrifugation Molecular weight Salting out Hydrophobicity of proteins decreases based on the solubility Dialysis Molecular size (Here, the smaller molecules move out of the membrane)  Protein Chemistry-2 Samreen Sheik, Department of Biochemistry Manipal University College Malaysia (MUCM) Learning Outcomes Protein structure Define the four levels of structural organization with examples Primary structure o Define the term peptide bond o Explain the properties of peptide bond o Mention the significance of few physiologically important peptides o Show the 'N' and 'C' terminals of a peptide o Illustrate with an example the nomenclature of peptides Secondary structure o Explain the structure of -helix, -pleated sheets and -bends with suitable diagrams, with the occurrence for each and the bonds stabilizing them o Describe with diagrams the various types of supersecondary structures Tertiary structure o Describe tertiary structure and the importance of domains o List the bonds/forces that stabilize tertiary structure Quaternary structure - List the bonds/forces that stabilize quaternary structure and list. examples of proteins with quaternary structure Proteins are the polymers of amino acids linked together by a peptide bond Monomeric protein: Protein with a single polypeptide chain.Eg: Keratin, Myoglobin Oligomeric protein: Protein with more than one single polypeptide chain. Eg: Hemoglobin, Lactate dehydrogenase The proteins are formed on the ribosomes in the linear form and then fold and attain a biologically active three-dimensional (3-D) conformation Proteins have four different levels of structural organization. Primary structure Secondary structure Tertiary structure Quaternary structure * A monomeric protein shows only up to tertiary structure. * Oligomeric proteins exhibit quaternary structure. Definition of levels of organization. 1. Primary structure of proteins indicates the number and sequence of amino acid residues from N-terminal to the C-terminal. Eg: Insulin 2. Secondary structure refers to the folding of the polypeptide chain, formed by the interaction between amino acids which are relatively close to each other in the primary structure of amino acids. Eg: Hemoglobin, Myoglobin, Keratin 3. Tertiary structure refers to the further folding of the secondary structure of the polypeptide chain to form functionally active three-dimensional conformation. 4. Quaternary structure refers to the spatial arrangement of the subunits of an oligomeric protein.Eg: Hemoglobin,Immunoglobulins and Lactate dehydrogenase has four globin chains. Primary structure Definition:----------------------- Features: Primary structure is maintained by strong covalent peptide bonds and hence are not affected during denaturation. The amino acids are arranged in a linear chain The primary structure of each protein has a unique amino acid sequence and numbers decided by the genes contained in DNA. The biological activity of the protein is dependent on the primary structure. A slight change in the primary structure of the proteins may result in the loss of biological activity of the protein. For example: A change in the 6th amino acid in the globin chain of haemoglobin (Glutamic acid replaced by Valine) causes Sickle Cell anaemia. Insulin as an example of primary structure peptide Insulin has two polypeptide chains, A and B. A chain (Glycine) has 21 amino acids, while the B chain (Phenylalanine) has 30 amino acids. The two chains are held together by 2 interchain disulfide bonds at A chain 7th cysteine and B chain 7th cysteine, A chain 20th cysteine and B chain 19th cysteine.There is intrachain disulfide bond between 6th and 11th cysteine residues of A chain.  Secondary structure -helix: -Is the most common spiral structure of protein. Features: 1. The  -helix is a right-handed tightly packed coiled structure with amino acid side chains extending outward from the central axis. 2. The  -helix is stabilized by extensive hydrogen bonding. It is formed between H atom attached to peptide N, and O atom attached to peptide C. The hydrogen bonds are individually weak but collectively, they are strong enough to stabilize the helix. 3. All the peptide bonds, except the first and last in a polypeptide chain, participate in hydrogen bonding. 4. Each turn of the helix contains 3.6 amino acids and travels a distance of 0.54 nm. The spacing of each amino acid is 0.15 nm. 5. alpha-Helix is a stable conformation formed spontaneously with the lowest energy. 6.Certainamino acids (particularly proline) disrupt the-helix. Large number of acidic (Asp, Glu) or basic (Lys, Arg, His) amino acids also interfere with 􀁄-helix structure. -helix: Diagram must show: 1.show hydrogen bond between carboxyl and amino groups are parallel to the backbone. 2. R group extended outward.  Secondary structure -sheet: i. -sheet is an extended structure where the polypeptide chains are arranged in sheets(in pleated or zig zag pattern) ii. Stabilized by hydrogen bond which are perpendicular to the peptide backbone (between carboxyl and amino groups ) iii. The adjacent beta-strands are arranged either antiparallel or parallel depending on the direction of the bonding chains. Secondary structure: -sheet If the bonding chains run in the opposite direction If the bonding chains run in the same direction Eg:Fibroin of silk Eg:Flavodoxin  -bends (-turns)-Minor secondary structure β-Bends refers to the segment that connects the ends of adjacent strands of anti-parallel β pleates. They are composed of four amino acids, one of which may be proline/ Glycine, the amino acid that causes a kink in the polypeptide chain β-Bends are stabilized by the formation of hydrogen bonds between the first and the 4th amino acid residue resulting in firm 180o turn in the bend.Hence it is also called as reverse turn/hair pin turn Tertiary structure Definition------------- Features: refers to the foldings of the amino acids which are far apart from each other in linear primary structure,bought closer. Results in the orientation of hydrophobic side chains towards the interior and hydrophilic side chains towards the exterior on the surface of proteins. Results in the formation of Domains, are the structurally connected but functionally independent units of a protein that perform a particular function such as binding with substrate/ligands in the enzymes etc. Quaternary structure Bonds/Forces that stabilizes tertiary/Quaternary structure 1. Hydrophobic bonds(nonpolar side chains) 2. Hydrogen bonds (between polar groups) 3. Ionic bonds (-COO- with –NH3+) 4. Van der Waals forces 5. Disulfide bonds: covalent linkage from – SH (sulfhydryl group) of 2 cysteine to form cystine residue  Protein Chemistry-3 Samreen Sheik,Department of Biochemistry Manipal University College Malaysia (MUCM) Learning Outcomes Protein denaturation ✓ Define ✓ List the denaturing agents ✓ Explain its effects on the properties of a protein Describe the role of protein misfolding in amyloid disorders (with emphasis on Alzheimer’s disease) and prion disease Protein Denaturation Denaturation is the process of disorganization of native protein structure, which involves the loss of secondary, tertiary and quaternary structures without breaking the primary structure Denaturing agents i. Physical agents: Heat,UV radiation,X-ray,Ultrasound,Pressure. ii. Chemicalagents: Acids, Alkalies, Organic Solvents(ether,alcohol), Urea, Salicylate,heavy metals(lead,mercury etc). Effect of denaturation on protein i.Unfolding secondary, tertiary, quaternary structure. ii. Primary structure remain intact. iii. Decreases the solubility. iv. Increases the precipitability. v. Causes loss of biological activity. vi.Are easily digestible. Protein Folding Protein folding is a process by which a polypeptide chain folds to become a biologically active protein in its native 3D structure. Protein structure is important and is linked to its function. Protein folding is a very sensitive process that is influenced by several external factors including electric and magnetic fields, temperature, pH, chemicals, space limitation and molecular crowding. These factors influence the ability of proteins to fold into their correct functional forms. When a protein errors at folding it can cause denaturation of the protein. Denaturation is the loss of protein structure and function. Misfolding does not always result in a complete lack of function, but there is a partial loss of function. This miss functioning of proteins can lead to different diseases in the human body. The role of protein misfolding in amyloid disorders Mechanism of protein misfolding 1. Misfolding of proteins may occur spontaneously or caused by a mutation in a particular gene. 2. Chaperones (Heat shock proteins, HSP) monitor the quality of the folded chains and they can unfold and refold the misfolded proteins. 3. But if the misfolded proteins does not get corrected by chaperons they undergo degradation by protease enzymes. 4. Still, if the misfolded proteins remain undegraded , they aggregate and accumulate in the human body 5. This causes toxic effects by binding and sequestering other cytosolic proteins. 6. For example, the formation of amyloid plaques in Alzheimer’s disease and infectious prion proteins in Prion disease. Alzheimer Disease It is a degenerative disease-causing loss of neurons in the cortex of the brain Most common cause of Dementia *poor memory *difficulty learning Occurs mainly due to plaques and tangles Accumulation of plaques In the cell membrane of brain, the protein called Amyloid Precursor Protein(APP) is present which is responsible for the cell repair. Normally α- secretase and γ-secretase are involved in the proteolytic cleavage and the formed subunits are soluble and functional. In an abnormal proteolytic cleavage β- secretase form long,fibrillar,insoluble and aggregating proteins called β Amyloid plaques. The plaques misinterpret neuron signalling and relay information and thus brain function gets impaired. Accumulation of tangles Neurons are held together by microtubules. A special protein called tau(τ) protein stabilizes the microtubules. Beta-amyloid plaques formed around the neuronal cells initiate the signaling pathways inside the neuronal cells leading to the dysfunctional tau protein, which gets tangled leading to neurofibrillary tangles and become dysfunctional. Prion Disease PrP:Gene encoding for Prion Protein PrPc : Normal Cellular Prion Protein Present on the surface of cells in neurons and are used during synapses PrPsc : Abnormal prion protein which occurs due to the misfolding of the protein This leads to diseases called Creutzfeldt-Jakob disease and Kuru in humans Scrapie disease in Sheep's Mad cow disease/bovine spongiform encephalopathy (BSE) in cattle