Proteins 2 Lecture Notes PDF

PROTEINS 2 By Inonda RN  Peptides Are polymers of amino acids Biologically occurring polypeptides range in size from small to very large, consisting of two or three to thousands of linked amino acid residues. Two amino acid molecules can be covalently joined through a...

PROTEINS 2 By Inonda RN  Peptides Are polymers of amino acids Biologically occurring polypeptides range in size from small to very large, consisting of two or three to thousands of linked amino acid residues. Two amino acid molecules can be covalently joined through a peptide bond, to yield a dipeptide Condensation and Hydrolytic Reactions Figure  dehydration reaction removes a hydroxyl group from the carboxyl end of one amino acid and a hydrogen from the amino group of another. This is acovalent The resulting condensation reaction bond is called a peptide bond.  Polypeptide backbone is the repeating sequence of the N-C-C- N-C-C… in the peptide bond The side chain or R group is not part of the backbone or the peptide bond  In a peptide, the amino acid residue at the end with a free -amino group is the amino-terminal (or N- terminal) residue; the residue at the other end, which has a free carboxyl group, is the carboxyl-terminal (C- terminal) residue.  Nomenclature Amino acid residues in polypeptides are named by dropping the suffix - ine, in the name of the amino acid and replacing it by -yl. Naming starts from the amino terminal to the carboxyl terminal with the last amino acid bearing the full name of the parent amino acid eg glycyl-alanyl-tyrosine.  Biologically Active Peptides Oxytocin: 9 amino acid residues ( gly-leu-pro-cys-asn-gln-ile-tyr- cys) Glutathione: 3 amino acid residues(glu-cys-gly) Insulin: 51 amino acid residues Glucagon: 29 amino acid residues Adrenocorticotrophic hormone: 39 amino acid residues GLUTATHIONE  Classification Peptides are classified depending on the number of amino acid residues they have. Dipeptide……2 Tripeptide 3 Nanopeptide 9 etc Oligopeptide more than 10 to 20 Polypeptide More than several dozens  Classification Proteins can further be classified depending on :- Composition Shape Function Solubility  Functions 1.Biological catalysts- enzymes 2. Hormones eg insulin, glucagon 3. Neurotransmitters eg glycine 4. Defense eg antibodies 5. Transport in body eg albumin, hemoglobin 6. Locomotion eg actin, myosin 7. Blood clotting eg thrombin and fibrinogen 8. Structural: Collagen, elastin and keratin.  Levels of Organization Primary structure – Amino acid sequence of the protein Secondary structure – H bonds in the peptide chain backbone -helix and -sheets Tertiary structure – Non-covalent/ covalent interactions between the R groups within the protein Quanternary structure – Interaction between 2 polypeptide chains  PRIMARY STRUCTURE The primary structure of a protein is its unique sequence of amino acids. – Lysozyme, an enzyme that attacks bacteria, consists of a polypeptide chain of 129 amino acids.  A slight change in primary structure can affect a protein’s conformation and ability to function. In individuals with sickle cell disease, abnormal hemoglobins develop because of a single amino acid substitution. – These abnormal hemoglobins crystallize, deforming the red blood cells and leading to clogs in tiny blood vessels. The most abundant amino acids in proteins are Leu, Ala, Gly, Ser, Val, and Glu; the rarest are Trp, Cys, Met, and His. The characteristics of an individual protein depend more on its amino acid sequence than on its amino acid composition  SECONDARY STRUCTURE The secondary structure of a protein results from hydrogen bonds at regular intervals along the polypeptide backbone. – Typical shapes that develop from secondary structure are coils (an alpha helix) or folds (beta pleated sheets).  SECONDARY STRUCTURE The structural properties of silk are due to beta pleated sheets. – The presence of so many hydrogen bonds makes each silk fiber very strong  Protein Folding 2 regular folding patterns have been identified – formed between the bonds of the peptide backbone -helix – protein turns like a spiral – fibrous proteins (hair, nails, horns) -sheet – protein folds back on itself as in a ribbon –globular protein  Features of α- helix Backbone tightly wound along an axis Each helix contains 3.6 a acid residues/ turn with R groups of amino- acids projecting outwards. Stabilized by hydrogen bonds between- N-H and C=O that are 4 residues back ( ie NH group of 6th peptide bond is hydrogen bonded to C=O of 2nd peptide bond.  Each peptide bond participates in hydrogen bonding Present in most fibrous tissues where the helix is right-handed. More stable Have intrachain hydrogen bonds Aromatic amino acids , charged and hydrophobic amino acids destabilize chain if not well spaced.. Glycine if closely together cause coiling of the helix Proline terminates helix by causing a kink.  Features of β- helix Polypeptide chain fully extended to form a sheet. 2-5 chains arranged side by side Can have chains running in same direction N to C terminal. Parallel β- pleated sheet Can have chains running in opposite directions. Anti-parallel β-pleated sheet. Stabilized by interchain hydrogen Anti-parallel chains  Tertiary proteins Tertiary structure is determined by a variety of interactions among R groups and between R groups and the polypeptide backbone. – These interactions include hydrogen bonds among polar and/or charged areas, ionic bonds between charged R groups, and hydrophobic interactions and van der Waals interactions among hydrophobic R groups.  Quarternary structure This results from the aggregation of two or more polypeptide subunits. – Hemoglobin is a globular protein with two copies of two kinds of polypeptides. – Lactate dehydrogenase , creatine phosphokinase.  Protein Folding The peptide bond allows for rotation around it and therefore the protein can fold and orient the R groups in favorable positions Weak non-covalent interactions will hold the protein in its functional shape – these are weak and will take many to hold the shape  Bonds involved in protein folding Hydrophobic interactions Hydrogen bonds Electrostatic interactions ( salt linkages) Van der waals forces Non-covalent Bonds in Proteins  Proteins at Work The conformation of a protein gives it a unique function To work proteins must interact with other molecules. Ligand – the molecule that a protein can bind Binding site – part of the protein that interacts with the ligand – Consists of a cavity formed by a specific arrangement of amino acids Ligand Binding  Formation of Binding Site The binding site forms when amino acids from within the protein come together in the folding The remaining sequences may play a role in regulating the protein’s activity  Protein Folding Proteins shape is determined by the sequence of the amino acids The final shape is called the conformation and has the lowest free energy possible(most stable) Denaturation is the process of unfolding the protein – Can be done with heat, pH or chemical compounds – If the chemical compound can be removed the protein can renature or refold  Denaturing Alteration of the protein’s shape and thus functions through the use of – Heat – Acids – Bases – Salts – Mechanical agitation Primary structure is unchanged by denaturing A protein’s conformation can change in response to the physical and chemical conditions. Changes in pH, salt concentration, temperature, or other factors can unravel or denature a protein. These forces disrupt the hydrogen bonds, ionic bonds, and disulfide bridges that maintain the protein’s shape. Some proteins can return to their functional shape after denaturation, but others cannot, especially in the crowded environment of the cell. Usually denaturation is permanent  Stabilizing Cross-Links Cross linkages can be between 2 parts of a protein or between 2 subunits Disulfide bonds (S-S) form between adjacent -SH groups on the amino acid  Clinical importance of denaturation This is key in determining the structure of a given protein eg blood constituents, enzymes etc. It separates/ unfolds the protein into its constituent amino acids( primary structure) and it’s the first step in protein sequencing. Basis of sterilization. First step of digestion of proteins.  Types of proteins( Some proteins contain chemical composition) other than amino acids groups Conjugated proteins: Are proteins that contain a permanently associated chemical component in addition to amino acids. The non-aminoacid gp is PROSTHETIC GROUP Conjugated proteins are classified on the basis of the chemical nature of their prosthetic groups eg glycoproteins, lipoproteins and metalloproteins.  Types of Proteins( shape) Globular Proteins – – Compact shape like a ball with irregular surfaces – Enzymes are globular Fibrous Proteins – usually span a long distance in the cell – 3-D structure is usually long and rod shaped – Keratin and collagen are fibrous  Globular Proteins The side chains will help determine the conformation in an aqueous solution  Fibrous proteins Are secondary proteins Have α helix and β helix α helix was the first ordered structure of proteins to be discovered. Eg α keratin in hair and nails, collagen and elastin. β helix was the second ordered structure. Found in silk and spider web. Have β sheets. Provide support, shape and external protection.  Strength of fibrous proteins is enhanced by covalent cross-links between adjacent chains Mainly strengthened by disulphide bonds In toughest keratins, a high % of residues are cysteine.  CALCULATIONS The number of amino acid residues in a simple protein can be calculated as follows:- The average mol wt of a chain that has all the amino acids is 138 Consider that most of the residues are small, the average weight is 128 Consider that H2O is lost during peptide bond formation, average weight is 128- 18=110D 1. How many amino acid residues are in a chain that has a molecular weight of 50160D 2. A chain has 635 amino acid residues. Calculate its approximate molecular weight. 3. Calculate the actual weight of alanyl- glycyl-serine. 4. Draw alanyl-glycyl-serine indicating the peptide bonds. 5. A chain is made up of 2 tyrosine residues, 3 serine residues and 2 cysteine residues. Calculate its actual molecular weight. Analysis of protein mixtures.  Seperation of proteins Seperation of proteins depends on their 1. Solubility 2. Charge 3. Size 4. Shape 5. Affinity  Separation of proteins 1. Release of protein into solution – crude extract. Subjection of crude extract to fractionation by salting out which precipitates proteins Ammonium sulphate used.  Dialysis Separates proteins from solvents using the size of proteins. Extract placed in a bag made of a semi permeable membrane and suspended in buffer. Salts and buffer interchanged while proteins remain in bag Method used to remove ammonium sulphate from proteins  Chromatographic methods Partition molecules between two phases, one mobile and the other stationary For separation of proteins the stationary phase may be a sheet of filter paper (paper chromatography) or a thin layer of cellulose, silica, or alumina (thin- layer chromatography- TLC) Column Chromatography Stationary phase a column containing small spherical beads of modified cellulose, acrylamide, or silica whose surface typically has been coated with chemical functional groups. These stationary phase matrices interact with proteins based on their charge, hydrophobicity, and ligand- binding properties.  Procedure A protein mixture is applied to the column and the liquid mobile phase is percolated through it. Small portions of the mobile phase or eluent are collected as they emerge  Partition Chromatography Type of column chromatography Separation depends on the relative affinity of different proteins for a given stationary phase and for the mobile phase. The concentration of the solute in the two phases at equilibrium at a given temperature is known as the PARTITION COEFFICIENT Proteins that interact more strongly with the stationary phase are retained longer.  The length of time that a protein is associated with the stationary phase is a function of the composition of both the stationary and mobile phases. To get the behaviour of an amino acid/protein with a solvent, we calculate the retardation factor Rf Rf = Distance moved by amino acid Distance moved by solvent front  Size Exclusion Chromatography Separates proteins based on their size. This is a function of molecular mass and shape Also known as gel filtration  Size exclusion chromatography employs porous beads Proteins which are too large to enter the pores (excluded proteins) remain in the flowing mobile phase and emerge before proteins that can enter the pores (included proteins) Proteins thus emerge from a gel filtration column in descending order of their size.  Gel filtration chromatography  Absorption Chromatography The protein mixture is applied to a column under conditions where the protein of interest associates with the stationary phase so tightly Non-adhering molecules are first eluted and discarded. Proteins are then sequentially released by disrupting the forces that stabilize the protein-stationary phase complex  This is done by using a gradient of increasing salt concentration. The composition of the mobile phase is altered gradually so that molecules are selectively released in descending order of their affinity for the stationary phase.  Ion exchange chromatography In ion exchange chromatography, charged molecules bind to oppositely charged groups that are chemically linked to a matrix such as cellulose or agarose. Anions bind to cationic groups on anion exchangers, and cations bind to anionic groups on cation exchangers. The most frequently used anion exchanger is a matrix with attached diethylaminoethyl (DEAE) groups, and the most frequently used cation exchanger is a matrix bearing carboxymethyl (CM) groups.  The binding affinity of a particular protein depends on the presence of other ions that compete with the protein for binding to the ion exchanger and on the pH of the solution, which influences the net charge of the protein.  Proteins with a net negative charge adhere to beads with positively charged functional groups, typically tertiary or quaternary amines (anion exchangers). Proteins compete against monovalent ions for binding to the support—thus the term “ion exchange.”  For example, proteins bind to diethylaminoethyl (DEAE) cellulose by replacing the counter-ions (generally Cl− or CH3COO−) that neutralize the protonated amine. Bound proteins are selectively displaced by gradually raising the concentration of monovalent ions in the mobile phase sequential elution of proteins may be achieved by changing the pH of the mobile phase Electrophoresis  Electrophoretic techniques are based on the movement of ions in an electrical field. Positively charged amino acids move towards the cathode while negatively charged a. acids move towards the anode Uncharged a acids remain at the point of application.  Importance of protein sequencing Can confirm effects of mutation Key in synthesis of vaccine by knowledge of proteins on viral membrane. Mechanisms of enzyme catalysis known. Key in DNA cloning.  Quantitative determination of proteins UV absorbtion @ 280nm ( Not very accurate since DNA absorbs here too) Biuret’s test : Detects peptide bonds Folin reagent : Detects tyrosine residues  Sequencing Edman’s degradation used Sequences from N- terminal Uses phenylisothiocyanate to bind the N amino acid Can cleave chain to smaller portions using:- Trypsin: Lysine or Arginine at C terminal Pepsin: phe, trp, Tyr (N) Cyanogen bromide: Met (C )