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Presentation on protein chapter of Biochemistry and Molecular Biology lecture

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  1. 1. Protein Course Title: Biochemistry and Molecular Biology Course No. PHR 202 Course Teacher: Shahana Sharmin Department of pharmacy, BRAC University
  2. 2. Bonds in Protein • Peptide bond: In protein , the α- carboxyl group of one amino acid is joined to the α-amino group of another amino acid by peptide bond with removal of water. It is also called an amino bond. • Many amino acids joined by peptide bonds form a polypeptide chain, which is not branched. • An amino acid in a polypeptide is called a residue. • The sequence of amino acids in a polypeptide chain is written starting with the amino terminal residue. Thus in the tripeptide Ala-Gly-Try(AGW), alanine is the amino terminal residue and tryptophan is the carboxy terminal residue. • A polypeptide chain consists of a regularly repeating part, called the main chain and a variable part comprising the distinctive side chain. The main chain is sometime called backbone.
  3. 3. Peptide bond is rigid and planer • Pauling and Corey found that peptide unit is rigid & planner. Resonance stabilization of the bond between the carbonyl carbon & the nitrogen atom of the peptide unit confers partial double bond character and hence rigidity on the C-N bond. There is no freedom of rotation about the bond. • Consequently all four atoms of the peptide unit lie in the same plane. The length of the peptide bond is 1.32A, which is between that of C-N single bond (1.49A) & C=N double bond (1.27A). • In contrast, the link between the α-carbon & the peptide nitrogen & the bond between the α- carbon & the carbonyl are pure single bond. Consequently there is a large degree of rotational freedom about these bonds on either side of the rigid peptide unit. The rigidity of the peptide bond enables protein to have well defined 3D forms. The freedom of rotation on either side of the peptide unit is equally important because it allows proteins to fold in many different ways.
  4. 4. Bonds in Protein… • Disulfide bond: In addition to peptide bonds, covalent disulfide bonds can form between cysteine residues by forming a sulfide bridge (-S-S-) formed from the same or different polypeptides. The product is a cysteine residues. • The cross links are formed by the oxidation of two cystein residues. • Intercellular proteins usually lack disulfide bonds, whereas extracellular proteins often contain several. • Importance of disulfide bond: • Without it protein has no pharmacological or enzymatic activities. • This bond confer additional stability to specific confirmations of proteins such as enzyme(e.g. Ribonuclease) and structural proteins (e.g.. Keratin). • It is important for the 3D structure of proteins for performing the function. Figure : The disulfide bond between two cysteine residues.
  5. 5. Amino Acid • Amino acids are molecules containing an amine group, a carboxylic acid group and a side- chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen. • An alpha-amino acid has the generic formula H2NCHRCOOH, where R is an organic substituent, the amino group is attached to the carbon atom immediately adjacent to the carboxylate group (the α–carbon).
  6. 6. Proteins are built of 20 amino acids
  7. 7. Proteins are built of 20 amino acids • Amino acids with aromatic side chain: Phenylalanine (F) Tyrosin(Y) Tryptophan(W) • Remarks: – All the three have π electron clouds. – Phenylalanine & Tryptophan are hydrophobic but Tyrosin is hydrophilic. – Tryptophan contains an indole ring. • Amino acids containing sulfur atoms: Cysteine (C) Methionine (M) • Remarks: – Both amino acids are hydrophobic. – Cystein contains a sulfhydral group (-SH) & Methionine contains a sulfur atom in threonine linkage (-S-CH3)
  8. 8. Proteins are built of 20 amino acids • Amino acids containing aliphatic hydroxyl groups: Serine(S) Threonine (T) • Remarks: – Both of them are much more hydrophilic and reactive. • Amino acids containing basic side chain: Lysine (K) Arginine (R) Histidine (H) • Remarks: – All are hydrophilic . – Lysine and Arginine are positively charged at neutral pH but Histidine can be uncharged or positively charged depending on its local environment. – Histidine is often found in the active sited of enzymes, where its imidazole ring canreadily switch between those states to catalyze the making and breaking bonds.
  9. 9. • Amino acids containing acidic side chains and their amide derivatives: Aspartate (D) Glutamate (E) Asparagine (N) Glutamine (Q) • Remarks: – Uncharged derivatives of Aspartate and Glutamate are Asparagine and Glutamine, which contain a terminal amide group in place of a carboxylate. Proteins are built of 20 amino acids
  10. 10. Protein have unique amino acid sequence • In 1953, Fredrick Sanger determined the amino acid sequence of Insulin, a protein hormone. This work is a landmark in biochemistry because it showed for the first time that a protein has a precisely defined amino acid sequence. • Sanger’s approach was first to separate the two polypeptide chain A & B of insulin & then to convert them by specific enzymatic cleavage into smaller peptides that contained regions of overlapping sequence. Using 1-fluoro-2,4- dintrobenzine (Sanger’s reagent) he removed & identified one at a time, the amino terminal residues of these peptides.
  11. 11. Protein have unique amino acid sequence…. • By comparing the sequence of overlapping peptides, he deduced an unambiguous primary structure of both the A & the B chain. • Now the complete sequence of more than 1000 proteins are known. The sequence of nucleotides in RNA, which in turn specifies the amino acid sequence of a protein. In particular, each of 20 amino acid is encoded by one or more specific sequences of three nucleotides. Furthermore, proteins in all organisms are synthesized from their constituent amino acids by a common mechanism.
  12. 12. Importance of amino acid sequence • Amino acids sequences are important for several reasons • Knowledge of the sequence of a protein is very helpful indeed essential in elucidating its mechanism of action. • Analysis of relations between amino acid sequence & 3D structure of proteins are uncovering the rules that govern the folding of polypeptide chains. Amino acid sequence is the link between the generic message in RNA & 3D structure that performs a proteins biological function. • Sequence determination is part of molecular pathology. Alterations in amino acid sequence can produce abnormal function & disease, such as sickle cell anemia & cystic fibrosis. • The sequence of a protein reveals much about its evolutionary history. Protein resemble one another amino acid sequence only if they have a common ancestor. Consequently molecule events in evolution can be traced from amino acid sequences.
  13. 13. Alpha helix • The alpha helix is one of the most common secondary structures in proteins. Amino acid side chains project outwards from the polypeptide backbone that forms the core of the helix. • The chain is stabilized in this conformation by hydrogen bonds between the backbone amino group of one amino acid and the backbone carbonyl group of another amino acid that is four positions away. • These interactions do not involve side chains. Thus many different sequences can adopt an a helical structure. • Alpha helices are regular cylindrical structures. • One full turn occurs every 3.6 residues and extends the length of the helix by approximately 0.5 nm. • Types: • The screw sense of a helix can be right handed (clockwise) or left handed (counterclockwise), the α-helices found in protein are right handed. • The α-helical content of proteins ranges widely, from nearly none to almost 100%. For example – – The digestive enzyme chymotripsin is virtually devoid of α-helix. – In contrast, about 7.5% of myoglobin and hemoglobin is α-helical.
  14. 14. Animation of Alpha helix Alpha helix.mp4
  15. 15. Coiled-coil structure of Alpha helix: Super helix • Two or more α-helices can entwine to form very stable structures, which can have a length of 100AO or more. These structures are called coiled-coil structure of α-helix. Such α- helical coiled coils are found in myosin & tropomyosin in muscle, fibrin in blood clots & keratin in hair. • 3-5% amino acids are responsible for coiled- coil structure. * Why the elucidation of a structure of a α-helix is a landmark in molecular biology? The elucidation of structure of α-helix is a landmark in molecular biology because it demonstrated that the confirmation of a polypeptide chain can be predicted of the properties of its compounds are rigorously & precisely known.
  16. 16. Beta pleated sheet • The beta sheet is another common secondary structure. In contrast to an alpha helix, it is formed by hydrogen bonds between backbone atoms on adjacent regions of the peptide backbone, called beta strands. • These interactions do not involve side chains. Thus, many different sequences can form a beta sheet. • A beta sheet is a regular and rigid structure often represented as a series of flattened arrows. Each arrow points towards the proteins C-terminus. • In the example shown here the two middle strands run parallel—that is, in the same direction—whereas the peripheral strands are anti- parallel. • The amino acid side chains from each strand alternately extend above and below the sheet, thereby allowing each side to have distinct properties from the other. Beta sheets are usually twisted and not completely flat."
  17. 17. Animation of Beta pleated sheet Beta sheet.mp4
  18. 18. Difference between α-helix & β-pleated sheet • A polypeptide chain in a β-pleated sheet called β- strands, is almost fully extended rather than being tightly coiled as in the α-helix. • The axial difference in β-sheet between adjacent amino acids is 3.5 AO , in contrast with 1.5AO for the α-helix. • Another differences is that the β-pleated sheet is stabilized by hydrogen bonds between NH & CO groups in different polypeptide strands, whereas in the α-helix the hydrogen bonds are between NH & CO group in the same strand. • According to running directions β-sheet is classified as parallel & anti-parallel & according to screw, sense α- helix is clockwise & anticlockwise.
  19. 19. Beta-turn or hairpin turn • Most proteins have compact, globular shapes which can change direction frequently. The predominant type in cytosol changes direction of β-sheets are accomplished by the structure known as β-turn or β-bend that often connects the ends of two adjacent strands of anti-parrallel β-sheet. The essence of this hairpin turn is that the CO group of residue n of a polypeptide is hydrogen bonded to the NH group of residue n+3. Thus polypeptide chain can abruptly reverse its direction. They are also known as reverse turn or hairpin bend. Fig : Structure of β-turn. The NH & CO groups of residue 1 of the tetra-polypeptide shown here are hydrogen bonded, respectively, to the CO & NH groups of residue 4 which results in a hairpin turn)
  20. 20. Protein structure • According to the architecture protein have four structure levels. – Primary structure – Secondary structure – Tertiary structure – Quaternary structure • Primary structure: The linear sequence of amino acids in a whole protein is called primary structure of proteins. Understanding the primary structure of protein is necessary to diagnose or study certain disease which result in proteins with abdominal amino acid sequences. E.g. Alanin-Glycin-Leucine-Valine. • Secondary structure: It is the 3D structure of short (3 to 30 residue), adjacent segments of polypeptide into geometrically ordered units where the amino acids are located near to each other in the linear sequence. E.g. α-helix, β-sheet. • Tertiary structure: It is the entire 3D of whole protein where the amino acids are located close each other or far away from each other in linear sequence. It indicates the 3D space, how secondary structure features- helices, sheets, bends, turns & loops assemble to form domains & how three domain relate spatially to one another. • Quaternary structure: It is the combination of more than one polypeptide or protein that may be structurally identical or totally unrelated. The arrangement of these polypeptide subunits is called the quaternary structure of the protein. If there are two subunits the protien is known as dimeric, if there are three subunits, trimeric & if there are several subunits , multimeric. E.g. haemoglobin is trimeric which contains 4 polypeptide chains (two α chains & two β chains) Subunits are held together by non-covalent interactions. (E.g. H-bond, ionic bond, etc. )
  21. 21. Animation of Protein structure Protein Structure.mp4
  22. 22. Classification of amino acids according to their H-bond potentialities.. • According to H-bond potentialities, amino acids can be classified into following 3 groups – • Hydrogen bond donor: The side chains of Tryptophan & Arginine can serve as H- bond donors only. • Hydrogen bond donor & acceptor: Like the peptide unit itself, the side chains of Asparagine, Glutamine, Serine & Threonine can serve as hydrogen bond donors & acceptors. H donor group of Tryptophan H donor group of Arginine H acceptor & donor group of GlutamineH acceptor & donor group of Asparagine H acceptor H donor H donor H acceptor
  23. 23. Classification of amino acids according to their H-bond potentialities.. • Hydrogen bond donor and/or acceptors: The hydrogen bonding capabilities of Lysine (the terminal –NH2 group), Aspartic & Glutamic acid (the terminal COOH group), Tyrosin & Histidine vary with pH. The H-bonding modes of these ionizable residues are pH dependent. Protonated form of Aspartic Acid Ionized form of Glutamic Acid H acceptor H donor H acceptor
  24. 24. Protein possess 3D structure • Protein possess 3D structure. The unique dimensional structure of each polypeptide is determined by its amino acid sequence. Several types of interactions cooperate in stabilizing tertiary structures of protein. • Disulfide bond: A disulfide bond is a covalent linkage from the sulfahydral group (-SH) of each of two cystine residue to produce a cystine residue. When cystine residues perform covalent bonding in their side chains, the polypeptide chain (-S-S-) becomes folded. • Hydrophobic interactions: In an aqueous environment, protein fold is driven by the strong tendency of hydrophobic residues to be excluded from water. Note that water is highly cohesive & the hydrophobic groups are thermodynamically more stable when cluster in the interior of the molecule than when extended into the aqueous surroundings. The polypeptide chain therefore fold spontaneously so that its hydrophobic side chains are buried and its polar, charged chains are on the surface. Thus 3D structure formation is enhanced. • Presence of amino acid Proline: Proline has a cyclic structure which markedly present in protein structure. Proline reverse the dimension of the polypeptide chain and thus provides curved protein. • Hydrogen bonds: Amino acid side chains containing oxygen or nitrogen bound hydrogen can form hydrogen bonds with electron-rich atoms. For example – alcohol group of Serine & Threonine can form hydrogen bond with oxygen of carboxyl groups or carboxy groups of peptide bonds. Formation of hydrogen bonds enhance the formation and stabilization of three dimensional structure of protein.
  25. 25. Denaturation of protein • Denaturation of protein means destruction of 3D structure of native or wild protein. • A native protein is converted into a denatured protein by cleavage of disulfide bonds present in it. For e.g. – • Ribonuclease a single polypeptide chain consisting of 124 amino acid residues has four disulfide bonds. This four disulfide bonds can be cleaved reversibly by reducing them with reducing agents. There are two types of agnets – • Physical agent – Thermal Denaturation. – Interfaces. – Mechanical treatments. – Hydrostatic Pressure. – Irradiation. • Chemical agent – Aqueous Organic Compounds. – Effect of pH. – Changes in Dielectric constant. – Ionic strength.
  26. 26. Physical agent • Thermal denaturation : Rate of denaturation depends on the temperature. As Temperature is increased – Affect interactions of tertiary structure – Increased flexibility → reversible – H-bonds begin to break → water interaction – Increased water binding – Increased viscosity of solution – Structures different from native protein Upon cooling – Aggregation – Loss of solubility – Water content affects heat denaturation – Splitting of disulfide bonds – Chemical alterations of amino residues – Inter- or Intra- crosslinks • Interface: • Liquid-air or Liquid-liquid interfaces • If allowed at interfaces → Unfold • Depends on – Rigidity of the 3-D structure – Number and location of hydrophobic groups – Accelerated if applied energy to cause shear
  27. 27. Chemical agent • Aqueous Organic Compounds: • Urea and Guanidine salts  Disrupts H-bonds  Decrease hydrophobic interactions • Surface-active agents (SDS)  Disrupt hydrophobic interactions  Increase internal repulsive forces  Unfold • Reducing agents  Reduce disulfide crosslinks (β –mercaptoethanol) • Effect of pH: • Proteins at physiological pH When pH is lower than pI -- • Strong acid with high temperature causes deamination followed by Hydrolysis peptide bonds. When pH is higher than pI -- • Formation of fibers causes denaturation • Chemical modifications lead to denaturation • Very high pH at elevated temperature results in peptide hydrolysis.
  28. 28. Effects of pH
  29. 29. This agents have different mechanism of action. β-marcaptoethanol is used to reduce the disulfide bond present in a native protein and β- marcapto forms mixed disulfides with Cystein side chains. In the presence of excess amount of β-marcaptoethanol the mixed disulfides also are reduced, so that the final product is a protein in which the disulfides (Cysteins) are fully converted into sulfhydrals (Cystein) Mechanism of action of reducing agent for Denaturation…
  30. 30. • Mechanism of action of reducing agent: 2. When ribonuclease can not be readily reduced by β-marcaptoethanol at 370 C and pH 7, then the Proline is partly unfolded by agents such as Urea or Guanidine hydrochloride. These agents disrupt noncovalent interactions. Thus when ribonuclease is treated with β-marcaptoethanol in 8M urea, the product is fully reduced, the poly chain becomes devoid of enzymatic activity. In other words ribonuclease is denatured by this treatment. Mechanism of action of reducing agent for Denaturation…
  31. 31. Effects of Denaturation in the body Protein Denaturation results in the unfolding & disorganization of the protein structure, which are accompanied by hydrolysis of peptide bonds & finally protein loses its biological activity. Denaturation of protein hamper the following functions- 1. Specific protein molecules called enzyme loses their catalytic power & all chemical reactions in biological systems catalyzed by those enzymes are hampered. 2. Proteins e.g. – hemoglobin, myoglobin etc. can not transport molecules or ions. 3. Muscle contraction may be hampered. 4. Highly specific proteins like antibodies can not maintain the immune system. 5. Receptor proteins can not generate & transmit nerve impulses. 6. Improved digestibility 7. Increased intrinsic viscosity 8. Inability to crystallize
  32. 32. Renaturation of protein • During renaturation, all the disulfide, hydrogen, and hydrophobic bonds that stabilize the native conformation are re-formed. • Christian Anfinsen made a critical observation that the denatured ribonuclease, freed of urea and β- mercaptoethanol by dialysis, slowly regained enzymatic activity. • This prove that in suitable oxidizing condition, denatured reversible produce oxidsed protein. • For e.g. denatured ribonuclease is converted into native ribonuclease by the following process- – At first the denatured protein is freed from urea & β-mercaptoethanol by dialysis in dialysis bagskept in buffer. Then it is exposed to air for air oxidation of the sulfahydral groups in reduced ribonuclease. When the sulfhydral of the denatured enzyme become oxidised by air, the enzyme is sponteneaously refolded into a catalyticallly active form. – Detailed study shows that nearly all the original enzymatic activity is regained if the sufhydral are oxidized under properties of refolded enzyme will be virtually identical with those of the native enzyme.
  33. 33. Protein Purification • A large amount of protein in there native state are not pure. Several thousand protein have been purified in active from on the basis of some characteristics, such as – Size – Shape – Charge – Specific binding affinity • At each step in purification, the preparation is assayed for a distinctive property of the protein of interest (e.g. – enzymatic activity) to assess the efficacy of the procedure. There are various methods of purification of protein. They are - – Purification according to size : • Dialysis • Gel filtration chromatography. – Purification according to solubility: • Salting out – Purification based on charge: • Ion exchange chromatography – Purification based on affinity: • Metal-Chelate affinity chromatography
  34. 34. Protein purification according to size • Dialysis: • From a mixture of protein of different sizes, desired proteins can be separated from small proteins and other molecules by dialysis. The procedure is – – The protein mixture is taken in a dialysis bag made up of semi-permeable membrane, such as cellulose membrane with pores. – The bag is then kept in a buffer solution maintaining the pH of buffer solution to that pH at which the desired protein is stable. – Then the molecules having dimensions significantly greater than the pore diameter are retained inside the dialysis bag, whereas smaller molecules and ions traverse the pore of such a membrane and emerge in dialysate outside the bag. • We will not get 100% pure protein molecules by this method because the intermediate size molecules are also present in the dialysis bag with large molecules. • To make this process as accurate as possible, we must know the molecular weight of the desired protein to fix the molecular weight cut off. Figure: Dialysis. Protein molecules (red) are retained within the dialysis bag, whereas small molecules (blue) diffuse into the surrounding medium.
  35. 35. • Gel-Filtration Chromatography: • More discriminating separations on the basis of size can be achieved by the technique of gel-filtration chromatography . • The sample is applied to the top of a column consisting of porous beads made of an insoluble but highly hydrated polymer such as dextran or agarose (which are carbohydrates) or polyacrylamide. Sephadex, Sepharose, and Bio-gel are commonly used commercial preparations of these beads, which are typically 100 μm (0.1 mm) in diameter. • Small molecules can enter these beads, but large ones cannot. The result is that small molecules are distributed in the aqueous solution both inside the beads and between them, whereas large molecules are located only in the solution between the beads. Large molecules flow more rapidly through this column and emerge first because a smaller volume is accessible to them. • More larger quantities of protein can be separated by gel filtration chromatography than by gel electrophoresis but at the price of lower resolution. Protein purification according to size
  36. 36. Gel-Filtration Chromatography Figure : Gel Filtration Chromatography A mixture of proteins in a small volume is applied to a column filled with porous beads. Because large proteins cannot enter the internal volume of the beads, they emerge sooner than do small ones.
  37. 37. Protein purification according to solubility • Salting out: • The solubility of most proteins is lowered at high salt concentration. This effect is called salting out which is very useful. • The dependence of solubility on salt concentration differs from one protein to another. Hence salting out can be used to fractionate proteins. E.g. 0.8 M ammonium sulfate precipitates fibrinogen, a blood clotting protein, where as 2.4 M is needed to precipitate serum albumin. • Procedure: • At first protein mixture is added to a certain concentration of (NH4)SO4 salt and some ppt is formed. Thus protein mixture is added to different concentrations of salt. Different salt concentrations causes precipitation of different proteins. Precipitation of different proteins are collected and dissolved in buffer [Phosphate buffer – combination of KH2PO4 (acidic) and K2HPO4 (basic)]. Then by running them in SDS- PAGE with marker desired protein is detected. The salt concentration which caused precipitation of that desired protein is used for further purification of that desired protein. • Application: – It is used for purifying proteins. – It is used for concentrating dilute solution of protein. – It is used for fractionating protein.
  38. 38. • Ion-Exchange Chromatography: • Proteins can be separated on the basis of their net charge by ion-exchange chromatography. If a protein has a net positive charge at pH 7, it will usually bind to a column of beads containing carboxylate groups, whereas a negatively charged protein will not . • A positively charged protein bound to such a column can then be eluted (released) by increasing the concentration of sodium chloride or another salt in the eluting buffer because sodium ions compete with positively charged groups on the protein for binding to the column. • Proteins that have a low density of net positive charge will tend to emerge first, followed by those having a higher charge density. Positively charged proteins (cationic proteins) can be separated on negatively charged carboxymethyl- cellulose (CM-cellulose) columns. • Conversely, negatively charged proteins (anionic proteins) can be separated by chromatography on positively charged diethylaminoethyl-cellulose (DEAE-cellulose) columns. Protein purification based on charge
  39. 39. • Metal-Chelate Affinity Chromatography: • It is another powerful and general applicable means of purifying proteins. It is considered as the best method of protein purification. It is called one stop procedure. • In this procedure, there is a complex between Histidine-Imidazole ring(of protein) and metal. Under suitable conditions, high concentration of imidazole releases protein from the complex of metal-protein. This is a very specific method for purification of protein. • Different metals are used for the purpose of purification by this process, e.g. – nickel, cobalt, calcium, manganese, cadmium, zinc, copper etc. • Procedure: • Insulin is a protein which is purified by this method where about 95% pure insulin can be obtained. If this method is performed after dialysis, the yield will be 95%. • Collection of protein solution / mixture: • At first the desired protein is converted into histidine-tag-protein. For e.g., we will use histidine-tag-insulin which is obtained by cloing insulin into a plasmid containing histidine-tag DNA. Then it is inserted in E.coli and plasmid will produce insulin containing six histidine (the histidine-tag-insulin) Sonication and centrifugation of such E. coli will produce hundreds of proteins in the solution including histidin-tag-insulin. Protein purification based on affinity
  40. 40. • Then Ni-sulfate is poured into resin column where resin-Ni complex is formed. Protein solution is then poured into resin column and there will be a complex of resin-Ni-histidine tag insulin. No other protein binds with the complex and simply go through the column. • As nickel binds with the nitrogen of imidazole ring present in histidine the resin-Ni-histidine tag insulin forms a strong complex. Now, finally, very high concentrated imidazole ring is added to the column so that Ni binds with this imidazole and release the histidine-tag-insulin. Then this released histidine- tag-insulin is collected to perform dialysis so that excess imidazole becomes separated. Thus, 95% pure protein is obtained. • The actual insulin and modified histidine-tag-insulin have some biological activity. Protein purification based on affinity
  41. 41. Protein Separation • Electrophoresis: A molecule with a net charge will move in an electric field. This phenomenon is termed as electrophoresis. This offers a powerful means of separating proteins and other macromolecules, such as DNA and RNA. • Why the process is carried out in gel? Electrophoretic separations are nearly always carried out in gels (or on solid support such as paper) rather than in free solution, for two reasons: – Firstly, gels suppress connective currents produced by small temperature gradients, a requirement for effective separation. – Secondly, gels serve as molecular sieves that enhances separation. • Mobility of molecules in gel: – Molecules that are small compared with the pores in the gel readily through the gel. – Molecules much larger than the pores are almost immobile. – Intermediate-size molecules move through the gel with various degrees of facility. • Media of electrophoresis: • Polyacrylamide gels are choice supporting media for electrophoresis because – They are chemically inert and are readily formed by the polymerization of acrylamide. – Morever, their pore sizes can be controlled by choosing various concentrations of acrylamide and methylene-bis-acrylamide at the time of polymerization.
  42. 42. Formation of polyacrylamide gel Formation of polyacrylamide gel: Acrylamide reacts with Methylene-bis-acrylamide to form polyacrylamide gel where Pursulfate (S2O8 2- ) is converted into Sulfate free radical (2SO4 - ) which act as a catalyst. Figure: Formation of a Polyacrylamide Gel A three-dimensional mesh is formed by co- polymerizing activated monomer (blue) and cross- linker (red).
  43. 43. Gel elctrophoresis • Proteins can be separated largely on the basis of mass by electrophoresis in a polyacrylamide gel under denaturating conditions. • Procedure: • The mixture of protein is first dissolved in a solution of sodium dodecyl sulfate (SDS), an anionic detergent that disruptd nearly all noncovalent interactions in native proteins. • Marcaptoethanol or dithiothreitol is also added to reduce disulfide bonds. • Anion of SDS bind to main chains at a ratio about one SDS for every two amino acid residues, which gives a complex of SDS with a denatured protein a large net negetive charge that is roughly proportional to the mass of the protein. • The negetive charge acquired on binding SDS is usually much greater than the charge on the native protein. • The SDS complexes with the denatured proteins are then electrophorised on a polyacrylamide gel, typically in the form of a thin vertical slab. • The directyion of electrophoresis is from top to bottom. • Finally, the proteins in the gel can be visualized by staining them with silver or a dye such as Coomassic blue, which reveals a series of bands. • Radioactive labels can be detected by placing a sheet of X-ray film over the gel, a process called autoradiography. • Small proteins move rapidly through the gel, whereas large ones stay at the top, near the point of application of the mixture. • The mobility of most polypeptide chain under these conditions is inversely proportionate to the logarithm of their mass. • Thus proteins of different molecular weight are separated and from the bands or SDS-PAGE, molecular weight of different proteins determined.
  44. 44. Gel electrophoresis Figure : Polyacrylamide Gel Electrophoresis (A)Gel electrophoresis apparatus. Typically, several samples undergo electrophoresis on one flat polyacrylamide gel. A microliter pipette is used to place solutions of proteins in the wells of the slab. A cover is then placed over the gel chamber and voltage is applied. The negatively charged SDS (sodium dodecyl sulfate)-protein complexes migrate in the direction of the anode, at the bottom of the gel. (B)(B) The sieving action of a porous polyacrylamide gel separates proteins according to size, with the smallest moving most rapidly.
  45. 45. Gel electrophoresis Figure: Staining of Proteins After Electrophoresis. Proteins subjected to electrophoresis on an SDS- polyacrylamide gel can be visualized by staining with Coomassie blue. Figure :Electrophoresis Can Determine Mass The electrophoretic mobility of many proteins in SDS-polyacrylamide gels is inversely proportional to the logarithm of their mass.
  46. 46. Isoelectric Focusing • Proteins can also be separated electrophoretically on the basis of their relative contents of acidic and basic residues. • The isoelectric point (pl) of a protein is the pH at which its net charge is zero. At this pH, its electrophoretic mobility is zero because z in equation 1 is equal to zero. • For example, the pI of cytochrome c, a highly basic electron-transport protein, is 10.6, whereas that of serum albumin, an acidic protein in blood, is 4.8. Suppose that a mixture of proteins undergoes electrophoresis in a pH gradient in a gel in the absence of SDS. • Each protein will move until it reaches a position in the gel at which the pH is equal to the pI of the protein. This method of separating proteins according to their isoelectric point is called isoelectric focusing. • The pH gradient in the gel is formed first by subjecting a mixture of polyampholytes (small multicharged polymers) having many pI values to electrophoresis. • Application: Isoelectric focusing can readily resolve proteins that differ in pI by as little as 0.01, which means that proteins differing by one net charge can be separated.
  47. 47. Isoelectric Focusing Figure :The Principle of Isoelectric Focusing A pH gradient is established in a gel before loading the sample. (A) The sample is loaded and voltage is applied. The proteins will migrate to their isoelectric pH, the location at which they have no net charge. (B) The proteins form bands that can be excised and used for further experimentation.
  48. 48. Two-dimensional method/Combined method • Isoelectric focusing can be combined with SDS-PAGE to obtain very high resolution separations. • A single sample is first subjected to isoelectric focusing. • This single-lane gel is then placed horizontally on top of an SDS-polyacrylamide slab. • The proteins are thus spread across the top of the polyacrylamide gel according to how far they migrated during isoelectric focusing. • They then undergo electrophoresis again in a perpendicular direction (vertically) to yield a twodimensional pattern of spots. • In such a gel, proteins have been separated in the horizontal direction on the basis of isoelectric point and in the vertical direction on the basis of mass. • Importance: • By this combined method, we can separate thousnad of proteins. • We can purify protein molecules • Two proteins having same isoelectric point (pI) but different mole. Wt. can be determined. • Two proteins having different isoelectric point (pI) but same mole. Wt. can be determined.
  49. 49. Two-dimensional method/Combined method Figure :Two-Dimensional Gel Electrophoresis (A) A protein sample is initially fractionated in one dimension by isoelectric focusing as described . The isoelectric focusing gel is then attached to an SDS-polyacrylamide gel, and electrophoresis is performed in the second dimension, perpendicular to the original separation. Proteins with the same pI are now separated on the basis of mass. (B) Proteins from E. coli were separated by two- dimensional gel electrophoresis, resolving more than a thousand different proteins. The proteins were first separated according to their isoelectric pH in the horizontal direction and then by their apparent mass in the vertical direction.
  50. 50. Synthesis of Peptide • General consideration: • Peptides are synthesized by linking an amino group to a carboxyl group that has been activated by reacting it with a reagent such as dicyclohexylcarbadiimide (DCC). • Reagent: • Procedure: • The t-BOC amino acid is activated by DCC. • Then the amino acid reacts with another amino acid attached to resin. Here the attack of a free amino group on the activated carboxyl leads to formation of a peptide bond an the release of dicyclohexyl urea. • After formation of peptide, this t-BOC protecting group can bind subsequently removed by exposing the peptide to dilute acid which leave peptide bond intact. • Note: Here the t-BOC amino acid is used because if a single free amino group and a single free carboxyl group of the same amino acid can bind in presence of DCC which inhibit the formation of peptide.
  51. 51. Synthesis of Peptide
  52. 52. Protein synthesis by solid phase method • Peptide can be synthesized by a solid phase method derived by R. Bruce Merrifield. Amino acids are added stepwise to a growing peptide chain that linked to an insoluble matrix, such as polystyrene beads. • Procedure: • All reactions are carried out in a single vessel, which eliminate losses due to repeated transfers of products. • Anchoring: The carboxy terminal amino acid of the desired peptide sequence is first anchored to polystyrene beads. • Deprotection: The t-BOC protecting group of this amino acid is removed by treating with F3- COOH. • Coupling: The next amino acid (in the protected t-BOC form) and dicyclohexylcarbodiimide (the coupling agent) are added together. • After formation of peptide bonds, excess reagents and dicyclohexylurea are washed away, which leaves the beads with the desired dipeptide product. • Repitition: Additional amino acids are linked by repeating 2nd and 3rd step. • Release or cleavage: At the end of the synthesis, the peptide is released from the beads by adding hydrofluoric acid (HF), which cleaves the carboxyl ester anchor without disrupting peptide bonds. • Protective groups on potentially reactive side chains are also removed at this time. • Advantages: • The purification of the synthesized peptide is not required because the desired product at each stage is bound to beads that can be rapidly filtered and washed.
  53. 53. Peptide synthesis by solid phase method Fig: Sequence of steps in solid phase peptide synthesis, (1) Anchoring of the C-terminal amino acid (2)Deprotection of the amino terminus. (3)Coupling of the next residue, step (2) & (3) are repeated for each added amino acid, (4) The complete peptide is released from the resin.
  54. 54. Importance of synthesizing peptides of defined sequence 1. Synthetic peptides can serve as antigens to stimulate the formation of specific antibodies. For instance, as discussed earlier, it is often more efficient to obtain a protein sequence from a nucleic acid sequence than by sequencing the protein itself. Peptides can be synthesized on the basis of the nucleic acid sequence, and antibodies can be raised that target these peptides. These antibodies can then be used to isolate the intact protein from the cell. 2. Synthetic peptides can be used to isolate receptors for many hormones and other signal molecules. For example, white blood cells are attracted to bacteria by formylmethionyl (fMet) peptides released in the breakdown of bacterial proteins. Synthetic formylmethionyl peptides have been useful in identifying the cell-surface receptor for this class of peptide. Moreover, synthetic peptides can be attached to agarose beads to prepare affinity chromatography columns for the purification of receptor proteins that specifically recognize the peptides.
  55. 55. 3. Synthetic peptides can serve as drugs. Vasopressin is a peptide hormone that stimulates the reabsorption of water in the distal tubules of the kidney, leading to the formation of more concentrated urine. Patients with diabetes insipidus are deficient in vasopressin (also called antidiuretic hormone), and so they excrete large volumes of urine (more than 5 liters per day) and are continually thirsty. This defect can be treated by administering 1-desamino- 8-d-arginine vasopressin, a synthetic analog of the missing hormone. This synthetic peptide is degraded in vivo much more slowly than vasopressin and, additionally, does not increase the blood pressure. 4. Finally, studying synthetic peptides can help define the rules governing the three-dimensional structure of proteins. Importance of synthesizing peptides of defined sequence
  56. 56. • Edman deviced a mothod for labeling the amino-terminal residue and cleaving it from the peptide without disrupting the peptide bonds between the other amino acid residues. • Principle: The Edman degradation sequentially removes one residue at a time from the amino end of a peptide. • Mechanism: Phenylisothiocyanate reacts with the uncharged terminal amino group of the peptide to form a phenylthiocarbamoyl derivative. • Then under mildly acidic condition, a cyclic derivatives of the terminal amino acid is liberated, which leaves an intact peptide shortened by one amino acid. • The cylcic compound is a phenylthiohydantoin (PTH) – amino acid, which can be identified by chromatographic procedures. [Furthermore, the amino acid composition of the shortened peptide (Arg, Asp, Gly, Phe) can be compared with that of the original peptide. (Ala, Arg, Asp, Gly, Phe). • The difference between these analysis is one Alanine residue, which shows that Alanine is the amino-terminal residue of the original peptide. Edman Degradation (Protein sequencing)
  57. 57. The Edman Degradation. The labeled amino-terminal residue (PTH-alanine in the first round) can be released without hydrolyzing the rest of the peptide. Hence, the amino- terminal residue of the shortened peptide (Gly-Asp-Phe-Arg-Gly) can be determined
  58. 58. Edman Degradation Figure :Separation of PTH-Amino Acids PTH-amino acids can be rapidly separated by high-pressure liquid chromatography (HPLC). In this HPLC profile, a mixture of PTH-amino acids is clearly resolved into its components. An unknown amino acid can be identified by its elution position relative to the known ones.
  59. 59. Edman degradation • Edman degradation is carried out on a machine called a sequenator. In a liquid-phase sequenator, a thin film of protein in a spinning cylindrical cup is subjected to the Edman degradation. • The reagents and extracting solvents are passed over the immobilized film of protein, and the released PTH-amino acid is identified by HPLC. One cycle of the Edman degrdation is carried out in less than two hours. • Advantages: • Hundreds of proteins have been sequenced by Edman degradation of peptides derived from specific cleavages. • Disadvantages: • It is a demanding and time consuming process.
  60. 60. Amino Acid sequences are sources of many kinds of Insight • A protein's amino acid sequence, once determined, is a valuable source of insight into the protein's function, structure, and history. 1.The sequence of a protein of interest can be compared with all other known sequences to ascertain whether significant similarities exist.. If the newly isolated protein is a member of one of the established classes of protein, we can begin to infer information about the protein's function. For instance, chymotrypsin and trypsin are members of the serine protease family, a clan of proteolytic enzymes that have a common catalytic mechanism based on a reactive serine residue. If the sequence of the newly isolated protein shows sequence similarity with trypsin or chymotrypsin, the result suggests that it may be a serine protease. 2.Comparison of sequences of the same protein in different species yields a wealth of information about evolutionary pathways. For example, a comparison of serum albumins found in primates indicates that human beings and African apes diverged 5 million years ago, not 30 million years ago as was once thought. 3.Amino acid sequences can be searched for the presence of internal repeats. Such internal repeats can reveal information about the history of an individual protein itself.
  61. 61. 4.Many proteins contain amino acid sequences that serve as signals designating their destinations or controlling their processing. A protein destined for export from a cell or for location in a membrane, for example, contains a signal sequence, a stretch of about 20 hydrophobic residues near the amino terminus that directs the protein to the appropriate membrane. Another protein may contain a stretch of amino acids that functions as a nuclear localization signal, directing the protein to the nucleus. 5.Sequence data provide a basis for preparing antibodies specific for a protein of interest. Peptides with these sequences can be synthesized and used to generate antibodies to the protein. These specific antibodies can be very useful in determining the amount of a protein present in solution or in the blood, ascertaining its distribution within a cell, or cloning its gene . 6.Amino acid sequences are valuable for making DNA probes that are specific for the genes encoding the corresponding proteins. Knowledge of a protein's primary structure permits the use of reverse genetics. DNA probes that correspond to a part of the amino acid sequence can be constructed on the basis of the genetic code. Amino Acid sequences are sources of many kinds of Insight
  62. 62. Recombinant DNA Technology Has Revolutionized Protein Sequencing • Hundreds of proteins have been sequenced by Edman degradation of peptides derived from specific cleavages. A huge effort is required to elucidate the sequence of large proteins, those with more than 1000 residues. For sequencing such proteins, a complementary experimental approach based on recombinant DNA technology is often more efficient. • Long stretches of DNA can be cloned and sequenced, and the nucleotide sequence directly reveals the amino acid sequence of the protein encoded by the gene. Recombinant DNA technology is producing a wealth of amino acid sequence information at a remarkable rate. • With the use of the DNA base sequence to determine primary structure, there is still a need to work with isolated proteins. The amino acid sequence deduced by reading the DNA sequence is that of the nascent protein, the direct product of the translational machinery. • Cysteine residues in some proteins are oxidized to form disulfide links, connecting either parts within a chain or separate polypeptide chains. • Chemical analyses of proteins in their final form are needed to delineate the nature of these changes, which are critical for the biological activities of most proteins.
  63. 63. Protein synthesis 1. DNA unwinds 2. mRNA copy is made of one of the DNA strands. 3. mRNA copy moves out of nucleus into cytoplasm. 4. tRNA molecules are activated as their complementary amino acids are attached to them. 5. mRNA copy attaches to the small subunit of the ribosomes in cytoplasm. 6 of the bases in the mRNA are exposed in the ribosome. 6. A tRNA bonds complementarily with the mRNA via its anticodon. 7. A second tRNA bonds with the next three bases of the mRNA, the amino acid joins onto the amino acid of the first tRNA via a peptide bond. 8. The ribosome moves along. The first tRNA leaves the ribosome. 9. A third tRNA brings a third amino acid 10.Eventually a stop codon is reached on the mRNA. The newly synthesised polypeptide leaves the ribosome.
  64. 64. •DNA is a very long molecule that looks like a twisted ladder. •It is made up of 4 different subunits called nucleotides which can be arranged in any order •DNA has two strands. •The strands are stuck together by the complementary bases. •Adenine to Thymine A-T •Cytosine to Guanine C-G
  65. 65. It is the Sequence of bases that act like a code The sequence (order) of bases tells the cell what proteins to make. The sequence of bases dictates the sequence of amino acids, which determines the shape of a protein. If the protein is the wrong shape it will not work properly (it may work differently) So if the sequence in the DNA is wrong it may result in a genetic disease
  66. 66. Transcription 1 (making a mRNA copy of DNA) •The part of the DNA molecule (the gene) that the cell wants the information from to make a protein unwinds to expose the bases. •Free mRNA nucleotides in the nucleus base pair with one strand of the unwound DNA molecule.
  67. 67. Transcription 2 •The mRNA copy is made with the help of RNA polymerase. This enzyme joins up the mRNA nucleotides to make a mRNA strand. •This mRNA strand is a complementary copy of the DNA (gene) •The mRNA molecule leaves the nucleus via a nuclear pore into the cytoplasm
  68. 68. tRNA – pick up their specific amino acids from the cytoplasm
  69. 69. mRNA attaches to small ribosomal subunit
  70. 70. Translation - outline
  71. 71. Translation. mRNA used to make polypeptide chain (protein)
  72. 72. •First the mRNA attaches itself to a ribosome (to the small subunit). •Six bases of the mRNA are exposed. •A complementary tRNA molecule with its attached amino acid (methionine) base pairs via its anticodon UAC with the AUG on the mRNA in the first position P. •Another tRNA base pairs with the other three mRNA bases in the ribosome at position A. •The enzyme peptidyl transferase forms a peptide bond between the two amino acids. •The first tRNA (without its amino acid) leaves the ribosome.
  73. 73. Translation 2 The ribosome moves along the mRNA to the next codon (three bases). The second tRNA molecule moves into position P. Another tRNA molecule pairs with the mRNA in position A bringing its amino acid. A growing polypeptide is formed in this way until a stop codon is reached.
  74. 74. End of Translation A stop codon on the mRNA is reached and this signals the ribosome to leave the mRNA. A newly synthesised protein is now complete!
  75. 75. Animation of protein synthesis Protein Synthesis Animation Video.mp4
  76. 76. Additional ????? • Short note on: – SDS-PAGE – Iso-electric point – Native protein – Denatured protein – Zwitter ion • How α-amino acids are optically active? • What is the difference of proline from other amino acids? • Why protein possess three dimensional structure?
  77. 77. Reference book… • Biochemistry, 5th ed., J.M. Berg, J.L. Tymoczko and L. Stryer, Freeman (1995) (or an earlier edition). • Principles of Biochemistry – Leninger.