Protein Chemistry PDF

PROTEIN CHEMISTRY Dr PIYUSH B. TAILOR Associate Professor Depart. Of Biochemistry Govt. Medical College Surat.  INTRODUCTION Abnormality in protein structure will lead to major diseases with profound alterations in metabolic...

PROTEIN CHEMISTRY Dr PIYUSH B. TAILOR Associate Professor Depart. Of Biochemistry Govt. Medical College Surat.  INTRODUCTION Abnormality in protein structure will lead to major diseases with profound alterations in metabolic functions. Polymers of amino acids Linked by peptid bond  Amino acid: Basic unit of protein R NH3+ C COO- Amino group Carboxylic acid group H Different side chains, R, determin the properties of 20 amino acids.  Two amino acids = Dipeptide Three amino acids = Tripeptide Four amino acids = Tetrapeptide. < 10 amino acids together = Oligopeptide 10 -50 amino acids = Polypeptide. Even though there are 20 amino acids, by changing the sequence of combination of these amino acids, nature produces enormous number of markedly different proteins. Examples on Peptides: 1- Dipeptide Example: Aspartame acts as sweetening agent = aspartic acid + phenyl alanine. 2- Tripeptide Example: GSH – Reduce Glutathione = Glutamic acid + Cysteine + Glycine. = Helps in absorption of amino acids = Protects against hemolysis of RBC 3- octapeptides: (8 amino acids) Examples: Oxytocine and Vasopressin (ADH). 4- polypeptides: more than 10 amino acids: e.g. Insulin hormone  Type of Protein Structure 1. Primary 2. Secondary 3. Tertiary 4. Quaternary  Primary Structure. To write Amino acid Sequence of polypeptide chain Amino terminal on Left Carboxyl terminal on Right  Primary Structure.  Primary Structure & Functional Relationship Example 1. Sickle Cell Disease 2. Pre-pro-insulin to Pro-insulin to Insulin 3. Thallasemia Sickle Cell Disease Gene to Protein Sequence  Sickle Cell Disease Mutation In Gene for Beta chain of Haemoglobin – Adenine (A) is replace by Thymine (T) – In genetic codon = GAG converted to GTG During protein synthesis (transcription) – GAG represent Glutamic acid – GTG represent Valine Because of mutation, on 6th position in beta chain of haemoglobin, glutamic acid is replace by valine Normal Haemoglbin (HbA) is converted to abnormal sickle haemoglobin (HbS) – function get affected This is explain that – “ Sequence of A.A. get change protein function get change” `  Pre-pro-insulin to Pro-insulin to Insulin Pre-pro-insulin & pro-Insulin ,both are inactive Proinsulin – single chain with 84 amino acids – Inactive form Insulin – double chain (A & B chain) with 51 amino acids – Active form When Proinsulin is converted to insulin – Sequence & number of amino acid, both get change – So Function of protein get change – Protein (insulin) became non-functional to functional  Thalassemia Normal HbA = Alpha (141 A.A.) & Beta (146 A.A.) Chain Mutation – Non sense type of mutation (one of the etiology) – Premature termination of haemoglobin chain synthesis – Alpha or Beta either absent or short – Alpha or Beta thalassemia occur Number of amino acid get changed Functional protein (HbA) become non-functional Primary structural Functional Relation “Whenever Suquence or / and number of amino acid get change , protein function get change” Either Protein became Functional to Non-functional – HbA to Sickle cell disease (sequence of A.A. changed) – HbA to Thalassemia (Number of A.A. changed) Or Protein became non-functional to functional – Pro-insulin to Insulin (sequence & number of A.A. both changed) Recombinant Insulin Recombinant Insulin  At What pH This ion molecule has least solubility?  Extra 2 Arginine….Added in Human Insulin Is it make any change in charge? – More positive – More Negative – No any change What shell be added more to make it neutral (Net Charge Zero = Zwitter Ion )? – Positive ions (H+) – Negative ions (HCO3-) Can it change pI of insulin ? – Decrease – Increase – Unchange Extra 2 Arginine….Added in Human Insulin Human insulin has isoelectric pH of 5.4. what can be pI of this newly for recombinant insulin? – More than 5.4 – Less than 5.4 In human body, this new recomb-insulin is going to expose pH of _______. Recombinant insulin pI (6.7) is nearer to physiological pH (7.4) than human insulin pI (5.4). So what will be effect on it’s solubility? – More soluble – Less soluble (precipitation) Glargine Insulin (Lantus)  Primary Structure It is representing Numbers of Amino acids Sequence of Amino acids Unique amino acid sequence decided by the genes. Maintained by peptide linkages. This two things decide higher levels of organization (secondary, tertiary & quaternary) of protein.  Naming of Polypeptide To write A.A. Sequence of polypeptide chain Amino terminal on Left Carboxyl terminal on Right Each component A.A. in a polypeptide is called a “residue” In a polypeptide , all A.A. residues suffixes (-ine, -an, -ic, or -ate) changed to -yl, with the exception of the C-terminal amino acid. E.g. Tripeptide = N-terminal valine, glycine, & C- terminal leucine “valylglycylleucine”  Charecteristics of a peptide bond It’s a partial double bond. ‘trans’ in nature Freedom of rotation with limitation to “R” group Having rotation of 180 degree single bond = 360 degree double bond = 0 degree Distance is 1.32 A° single bond(1.42A°) double bond(1.27A°). Angles of rotation known as Ramchandran angles Separation of A.A. from Polypeptide Sequencing of the peptide from its N-terminal end Edman reagent = Phenylisothiocyanate Label N-terminal A.A. residue under mildly alkaline conditions. Phenylthiohydantoin (PTH) create instability in the N- terminal peptide bond It can be selectively hydrolyzed without cleaving the other peptide bonds Gene to Protein Sequence  Gene to Peptide sequence Has limitations – Not able to predict positions of disulfide bonds – Not able to identify any posttranslational modification. Therefore, direct protein sequencing is an extremely important tool. Branched and Circular proteins In linear polypeptide chains, there is Interchain Disulphide bridges. In different polypeptide chains Interchain = In same protein. Intrachain = Same polypeptide chain. Glutathione = Is it protein? Glutathione = Pseudopeptide  2. SECONDARY STRUCTURE Configurationally relationship between residues which are about 3-4 amino acids apart in the linear sequence. The structure is preserved by non-covalent forces or bonds. Disulfide bonds: =Covalent linkage between sulfhydryl group (–SH) =two cysteines may be far aways in the primary sequence or may even be different polypeptide chains; =contributes stability =Prevents denatured = E.g. Immunoglobulins Hydrophobic interactions Non polar side chains = Remain in the interior Polar side chains = Remain on the surface Hydrogen bonds Oxygen- or Nitrogen-bound hydrogen of side chains Alcohol groups of serine and threonine Can form hydrogen bonds with Oxygen of a carboxyl group of a peptide bond (electron-rich atoms) Ionic interactions Negatively charged groups, carboxyl group (–COO-) in the side chain of aspartate or glutamate. Positively charged groups, such as the amino group (–NH3+) in the side chain of lysine. Alpha Helix  1. Alpha helix spiral structure. Polypeptide bonds = Backbone. Side chain of amino acids extend outward. Stabilized by hydrogen bonds In each turn = 3.6 A.A. residues. Distance between each A.A. residue is 1.5A° Right handed. As A.A. in the proteins are of L-variety. Proline and hydroxyproline will not allow the formation of α-helix.  Example of Alpha Helix 1. Keratins = fibrous proteins in Hair & Nail It’s rigidity is determined by the number of disulfide bonds 2. Myoglobin = globular & flexible molecule. (Contrast to Keratin)  It can disturb Alpha helix Proline – Secondary amino group is not geometrically compatible – it inserts a kink in the chain Large numbers of charged amino acids – As it forms more ionic bonds or by electrostatically repelling each other. – E.g. glutamate, aspartate, histidine, lysine, or arginine Amino acids with bulky side chains or branched – Tryptophane, Valine, Isoleucine Beta Plated Sheet  2. Beta-pleated sheet The distance between adjacent a.a. is 3.5A°. It is stabilised by hydrogen bonds. Adjacent strands in a sheet Parallel Anti parallel E.g. Flavodoxin , Carbonic anhydrase Triple helical structure in collagen Flavodoxin  Beta Bands (beta turn) Reverse the direction of a polypeptide chain Helping it form a compact, globular shape. Found on the surface of protein molecules Supersecondary Structure (Motif) Combining of α-helices and β-sheets Form primarily the core region—that is, the interior of the molecule. Connected by loop β-bends Supersecondary structures are usually produced by packing side chains from adjacent secondary structural elements close to each other. Helix turn helix motif Zink finger Motif 3.TERTIARY STRUCTURE three dimentional structure of the whole protein. A.A. are far apart from each other in the linear sequence. But A.A. are close in the three dimention. It is maintained by non-covalent interactions hydrophobic bonds electrostatic bonds Van der Waals forces.  Domain Compact Globular Functional Unit of a protein. Independent region of the protein May be multiple = if protein has > 200 amino acids. Core of a domain is built from combinations of motifs. Folding of domain peptide chain is independently of folding in other domains.  Protein Folding Three-dimensional shape of the functional protein. Due to Interactions between A.A. side chains – Charges of a.a. side chains = attraction and repulsion – Hydrogen bonds – Hydrophobic interactions – Disulfide bonds Process of trial and error Tests many configuration This results in a correctly folded protein with a low- energy state  Denaturation of Protein Secondary, Tertiary and Quaternary structures of protein molecules broken down. Primary structure is not altered Unfolding & Disorganization of Protein Decreases the solubility So protein precipitated Loss of biological activity. Denatured proteins are re-natured when the physical agent is removed. (less possible) Factor which can does “Denaturation” Heating = Cooking, Heat coagulation test Urea = Renal Failure X-ray Ultra - violet rays High pressure = Cooking Organic solvent = Alcohol as antiseptic Metals = Mercury poisoning Acid & Alkali = Digestion  Chaperones = In protein folding Specialized group of proteins for the proper folding. Chaperones = “Heat Shock” proteins Information for Correct folding is in primary structure of protein. Interact with the polypeptide at various stages Some chaperones keeps protein unfolded until its synthesis is finished. Some Chaperones tends to fold so that their vulnerable, exposed regions do not become tangled(mixed). Some denatured protein can not be re-folded properly, – Because folding starts with stages its synthesis – Folding does not wait for synthesis be totally completed. 4.QUATERNARY STRUCTURE Certain polypeptides aggregate form one functional protein known as quaternary structure. The protein will lose its function when the subunits are dissociated. Stabilised by hydrogen bonds electrostatic bonds hydrophobic bonds van der Waals forces.  Depending on the number of the monomers Dimer(2) Tetramer(4). Each polypeptide chain is termed as Subunit or Monomer. For example, – Hemoglobin = 2 alpha and 2 beta chains – Immunoglobulin G = 2 heavy & 2 light chains – Creatine kinase is dimer – Lactate dehydrogenase is a tetramer.  Protein Misfolding Complex process Trial & Error process Misfolded proteins are usually tagged and degraded. Misfolding of proteins may occur – Spontaneously – By mutation in a particular gene Accumulation of misfolded proteins can cause disease E.g. Amyloidosis Amyloidosis  Amyloidosis = Alzheimer’s disease Accumulation of amyloid β (Aβ), 40 – 42 A.A. = “ Amyloids” Neurodegenerative disorder = “Alzheimer disease”. Found in the brain parenchyma & around blood vessels. Neurotoxic & leading to the cognitive impairment Abnormal proteolytic cleavage Formation of long fibrillar protein = β-pleated sheets. Second factor = Accumulation of neurofibrillary tangles Tangle protein = Role in assembly of the microtubular structure. Abnormal form of tangle protein = tau (τ) protein Abnormal neurofibrine actions  Prion Protein(PrP) & Prion disease PrP is a host protein. Present on the surface of neurons and Glial cells. Infective Prion Protein – No any change in amino acid and gene sequences – No change in primary structure differences – No any alternate post-translational modifications – changes in the three-dimensional conformation – number of α-helices noninfectious PrP (PrPc) are replaced by β-sheets in the infectious form(PrPsc). – highly resistant to proteolytic degradation – Accumulation of insoluble aggregates of fibrils Prion Protein  Prion Protein(PrP) & Prion disease The Prion Protein (PrP) as the causative agent of – Transmissible Spongiform Encephalopathies (TSEs) – Creutzfeldt Jakob disease in humans – Scrapie in sheep – Bovine spongiform encephalopathy in cattle Infective agent is thus an altered version of a normal protein Which acts as a “template” for converting the normal protein to the pathogenic conformation. TSEs are invariably fatal No treatment is currently available  Can dsRNA (double stranded RNA) & RISC (RNA Induce Silencer Complex) be useful to treat Prion Disease ?  CLASSIFICATION OF PROTEINS BASED ON FUNCTIONS a) Catalytic proteins = Enzymes b) Structural proteins = Collagen, Elastin c) Contractile proteins = Actin, Myosin d) Transport proteins = Hemoglobin, Myoglobin, Albumin, Transferrin. e) Regulatory proteins = ACTH, Insulin, Growth hormone. f) Genetic proteins = Histones g) Protective = Immunoglobing, Interferon, Clotting factors  Type of Protein based on Composition & Solubility Simple proteins (Only Polypeptide Chain) 1. Albumins 2. Globulins 3. Protamines More of arginine and lysine = Strongly basic E.g. protamine zinc insulin 4. Prolamines: Proline but lack in lysine. E.g. zein from corn, gliadin of wheat 5. Lectins: High affinity to sugar groups. human blood group A1 RBCs. 6. Scleroproteins: Form supporting tissues. E.g. Collagen = Cartilage & Tendon, keratin of hair,nail  Conjugated proteins (Protein + Non Protein group) 1. Glycoproteins: = Blood group antigens 2. Lipoproteins: = HDL, LDL,IDL,VLDL 3. Nucleoproteins: = Histones. 4. Chromoproteins = Hemoglobin , Flavoprotein 5. Phosphoproteins: = Casein of Milk , Vitellin of Egg yolk. 6. Metalloproteins: = Hemoglobin(iron), Cytochrome(iron), Tyrosinase(copper) , Carbonic anhydrase(zinc) Derived proteins Degradation products of the native proteins. Progressive hydrolysis of protein results in smaller & smaller chains protein→ peptones→ pepSdes→ amino acids  Type of Protein Based on Nutritional Value Nutritionally rich proteins: Complete proteins or First class proteins. Contains all the essential amino acids in required proportion. E.g. Casein of milk. Incomplete proteins: Lack one essential amino acid. Cannot promote body growth but able to sustain growth in adults. Proteins of Pulses = Deficient in Methionine Proteins of Cereals = Deficient in Lysine. If both are combined in the diet , adequate growth may be obtained. Poor proteins: Lack in many essential amino acids Zein of Corn lacks Tryptophan and Lysine.