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Protein- Protein configuration, Chemical bonds involved in protein structure, Ramachandran plot and Functions.

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  1. 1. PROTEIN STRUCTURE OF PROTEIN - Primary - Secondary - Tertiary and - Quaternary structures By, A.S. Ramya M.Sc. Microbiology SemII, LDC
  2. 2. AMINO ACID: Basic unit of protein
  3. 3. 20 DIFFERENT AMINO ACIDS • Nonpolar,hydrophobic • Polar, charged • Polar, uncharged
  4. 4. PROTEIN • Proteins are complex organic nitrogenous compound, which are large biomolecules, or macromolecules. • It is an essential part of all living organisms, especially as structural components of body tissues such as muscle, hair, wool etc., other proteins may be enzymes, hormones or oxygen carriers and antibodies. • Protein consisting of one or more long chains of amino acid residues. Proteins are made of 20 amino acids linked by peptide bonds.
  5. 5. • DISTRIBUTION OF AMINO ACID IN PROTEIN • The distribution of the 20 amino acid is not uniform in all proteins. • Eg. 40% of weight of fibroin and 25% by weight of collagen are accounted of glycine. • Human serum albumin with 585 amino acid residues has one tryptophan moiety. • LOCATION OF AMINO ACID IN PROTEIN • Amino acid with uncharged polar side chain are relatively hydrophilic and are usually on the outside of the protein, while the side on nonpolar or hydrophobic amino acids tend to cluster together on the inside. • Amino acids with acidic or basic chains are very polar, and they are nearly always found on the outside of the protein molecules.
  6. 6. PEPTIDE BOND • The amino acids units are linked together through the carboxyl and amino groups to produce primary structure of the protein chain .The bond between two adjacent amino acids is a special type of amine bond, known as peptide bond and the chain thus formed is called a peptide chain.
  7. 7. • N- AND C- TERMINALS- Each amino acid in the chain is termed a residue. The two ends of the peptide chain are named as amino terminal ( N- terminal ) and carboxyl terminal ( C- terminal). • These amino acid with free amino group is called N- terminal amino acid and one with free carboxyl group are called C- terminal amino acid. • The two terminal groups, one basic and another acidic are the only ionisable groups of any peptide chain except those present in the side chain.
  8. 8. • The four atoms (C, O, N, H) of the peptide bond form a rigid planar unit. There is no freedom of rotation about the C-N bond. On the contrary, the 2 single bonds on either side of the rigid peptide unit, exhibit a high degree of rotational freedom.
  9. 9. STEREOCHEMISTRY OF PEPTIDE CHAIN • All proteins are made up of L-configuration. This fixes steric arrangement at the α-carbon atom. The dimensions of the peptide chain are known to be 7.27A˚.
  10. 10. PROTEIN CONFIGURATION • Based on degree of complexity of the protein molecule it has categorized into 4 basic structural levels of organisation. • First defined by linderstrom-lang often referred to as primary, secondary , tertiary, quaternary structure.(1º, 2º, 3º, 4º) • 1º, 2º, 3º structural levels can exits in molecules composed of single polypeptide chain, whereas 4º involves interactions of polypeptides within a multichained protein molecule.
  11. 11. Primary structure (Amino acid sequence) consists of one or more linear chains of number of aminoacid units ↓ This unfold structure often assumes helical shape to produce secondary structure ( α-helix, β-sheet ) ↓ Tertiary structure ( Three-dimensional structure formed by assembly of secondary structures ) ↓ Quaternary structure ( Structure formed by more than one polypeptide chains, cetain protein has subunits (single protein molecule) interact with each other in a specific manner to form a protein complex. Multimeric structure of different monomer.
  12. 12. CHEMICAL BOND INVOLVED IN PROTEIN STRUCTURE • Protein synthesis is a multiple dehydration process. • PRIMARY BOND ( peptide bond, -CO-NH- ) – • The principal linkage found in all proteins is a covalent peptide bond. • Peptide bond is the backbone of the protein chain • It is a specialised amine linkage where C atom of –COOH group of one amino acid is linked with N atom of –NH2 group of the adjacent amino acid releasing a molecule of water (H2O). • This is a dehydration synthesis reaction (also known as a condensation reaction), and usually occurs between amino acids.
  13. 13. • SECONDARY BONDS- • Many of the properties of proteins do not coincide with the linear chain structure, thus indicating that a variety of bonds other then the peptide exist in them and they holding the chain in its natural configuration.
  14. 14. • 1. DISULFIDE BOND (-S-S-) • In addition to peptide bond, a second type of covalent bond found between amino acid residues in proteins and polypeptide in the disulfide bond, which is formed by the oxidation of the thiol or sulfhydryl groups (-SH) of two cysteine residues to yield a mole of cystine, an amino acid with a disulfide bridge.
  15. 15. • Disulfide Bond strength 50 kilocalories per mole and bond length of about 2A˚ between two sulfur atoms. • 2 cysteine residues located some distance apart in the polypeptide chain requires the polypeptide to folded back on itself to bring the sulfur groups close together. • Even a slight extension breaks the sulfide bond completely. They, therefore only stabilize the tertiary structure. • Eg . Insulin
  16. 16. • 2. HYDROGEN BOND ( >CO….HN< ) - A hydrogen bond is the electrostatic attraction between two polar groups that occurs when a hydrogen (H) atom covalently bound to a highly electronegative atom such as nitrogen (N), oxygen (O), experiences the electrostatic field of another highly electronegative atom nearby. • The formation of hydrogen bond is due to the tendency of hydrogen atom to share electrons with two neighbouring atoms O and N.
  17. 17. • For Eg. The carbonyl oxygen of one peptide bond shares its electrons with the hydrogen atom of another peptide bond. • An interaction sets in between a C=O group and the proton of an NH group if these groups come within a distance of about 2.8 A˚. • Hydrogen bonds are relatively weak linkages but many such bonds collectively exerts considerable force and help in maintaining the helical structure or secondary structure. • Eg. Keratin of wool, silk fibroin.
  19. 19. • 3. NONPOLAR OR HYDROPHOBIC BOND – • Many aminoacids (like alanine, valine, leucine, isoleusine, methionine, tryptophan, phenylalanine, tyrosine) have side chains or R groups which are essentially hydrophobic. • This R groups can unite among themselves with elimination of water to form linkages between the chains or between different chains. The assosiation of various R groups in this manner leads to a relatively strong bonding. • It also serves to bring together groups that can form hydrogen bonds or ionic bonds in the absence of water. • Each type of linkage helps in the formation of the other. • The hydrophobic bond also play an important role in other interactions. E.g. the formation of enzyme substrate complexes and antibody antigen reactions.
  20. 20. • 4. IONIC OR ELECTROSTATIC BOND – • Ions possessing similar charge repel each other whereas the ions having dissimilar charge attract each other. • Another instance of ionic bonding may be the interaction between the acidic and basic groups of the constituent amino acids. • Thus, ionic bonds between positively charged groups( side chains of lysine, arginine, histidine) and negatively charged aspartic and glutamic acids) do occur.
  21. 21. • When two oppositely charged groups are brought close together, electrostatic interactions lead to strong attraction, resulting in the formation of electrostatic bond. • In fact ionised group are more frequently found stabilising interactions between protein and other molecules. • Although these ionic bonds are weaker than the hydrogen bonds, are regarded as responsible for maintaining the folded structure (or tertiary structure) of globular protein.
  22. 22. PROTEIN ASSEMBLY • occurs at the ribosome • involves polymerization of amino acids attached to tRNA • yields primary structure
  23. 23. PRIMARY STRUCTURE • The primary structure of a protein refers to the linear sequence of amino acids in the polypeptide chain. • The main mode of linkage of the amino acids in the proteins is the peptide bond which links the α- carboxyl group of one amino acid residue to the α- amino group of the another.
  24. 24. RAMACHANDRAN PLOT • Ramachandran Plot is a way to visualize dihedral angles ψ against φ of amino acid residues in protein structure. Ramachandran recognized that many combinations of angles in a polypeptide chain are forbidden because of steric collisions between atoms. • TORSION ANGLE- A dihedral angle is the angle between two intersecting planes • PHI AND PSI ANGLES- The two torsion angles of the polypeptide chain, also called Ramachandran angles(after the Indian physicist who first introduced the Ramachandran plot), describe the rotations of the polypeptide backbone around the bonds between N-Cα (called Phi, φ) and Cα-C (called Psi, ψ).
  25. 25. • Peptide unit is a planar one. C=O and NH group of amino acid is lie in a plane. • With respect to the middle Cα the 2 peptide unit (C=O and NH) are oriented to one another. Thus, this Cα carbon atom connected with this 2 planar peptide unit. • Phi is the tortion angle about N-Cα • Psi is the tortion angle about Cα-C • The N-Cα and Cα –C has a single bond so it can be twisted or rotated. The tortional angle between two planes represent to what extent this groups are twisted.
  26. 26. • In this graph some areas are shaded means that some phi and psi combination is not allowed because of steric clashes of atoms are more and energetically unfavourable. • Shaded regions showing different types of clashes are posssible.
  27. 27. • Amino acid has own conformation in these phi and psi combination is represented by black spots • White regions are favourable regions. Black spots in these white regions assumes that this phi and psi combination represent good configuration of amino acids (i.e) helix, strand • Blue regions are allowed regions. • Any phi and psi values of a given sequence fall in the Ramachandran plot. • In shaded regions also some black points. Some reason like glycine have no side chain and it is highly flexible and number of clashes less, no unfavourable region, proline have special orientation, no unfavourable region.
  28. 28. • Less unfavoured and no clashes in the region are called outlayers. • Ramachandran plot validates the protein structure and guides the structure confirment. • Simplest and most sensitive means for accessing the quality of the protein model.
  29. 29. PROTEIN FOLDING • occurs in the cytosol • involves localized spatial interaction among primary structure elements, i.e. the amino acids • Therefore the protein can fold and orient the R groups in favorable positions • yields secondary structure • Weak non-covalent interactions will hold the protein in its functional shape – these are weak and will take many to hold the shape
  30. 30. • 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 • Denaturation is the process of unfolding the protein – Can be down with heat, pH or chemical compounds – In the chemical compound, can remove and have the protein renature or refold
  31. 31. REFOLDING • Molecular chaperones are small proteins that help guide the folding and can help keep the new protein from associating with the wrong partner.
  32. 32. SECONDARY STRUCTURE • If the peptide bond are the only type of linkage present in the proteins, these molecules would have behaved as irregularly coiled peptide chain of considerable length. • But the globular proteins have regular characteristic properties, indicating the presence of regular coiled structure in these molecules. • This secondary structure is non-linear and 3 dimensional. • Formed and stabilized by hydrogen bonding, electrostatic and vander Waals interactions • This involves folding of the chain which mainly due to the presence of hydrogen bonds. Thus, folding and hydrogen bonding between neighbouring amino acids results in the formation of a rigid and tubular structure called a helix.
  33. 33. • Based on nature of hydrogen bonding, two main types of secondary structure, the α-helix and the β-strand or β-pleated sheets, were suggested in 1951 by Linus Pauling and coworkers. • The unstretched protein molecules formed a helix (which are called the α-form) • The stretching caused the helix to uncoil, forming an extended state (which he called the β-form) • They have a regular geometry, being constrained to specific values of the dihedral angles ψ and φ on the Ramachandran plot.
  34. 34. α-helix • The α-helix is a rod like structure. Tightly coiled polypeptide chain forms the inner part of the rod, an the side chains extend outward in a helical array. • The α-helix is stabilised by hydrogen bonds between the NH and CO groups of the peptide chain, assumes a rod- like structure. • A very interesting feature of the helix, besides the periodicity, is the fact that the carbonyl group of every peptide bond is in position to form a hydrogen bond with the amine group of the peptide bond in the next turn of the helix.
  35. 35. • The amino acid residue in an α-helix have conformations with φ (phi) = -60º and ψ (psi)= -45º to -50º. • The has a pitch of 5.4A˚ and contains 3.6 amino acids per turn of helix , thereby giving rise per residue of 1.5A˚ • A α-helix can be right handed(clockwise) or left handed (anticlockwise). • Biologically functional proteins do not usually exhibit cent percent α helix structure. Some have a high percentage of their residues in helical structure. e.g. hemoglobin, myoglobin; others have low percentage e.g. cytochrome C, chymotrpsin.
  36. 36. ᵦ-pleated sheet • ᵦ-pleated sheet in which the peptide chain is bent to form zig-zag conformation. • The pleated sheet structure depends on intermolecular hydrogen bonding, although intramolecular hydrogen bonds are also present. • The pleated sheet structure is formed by the parallel alignment of a number of polypeptide chain in a plane, with hydrogen bonds between the C=O and –N-H groups of adjacent chains.
  37. 37. • In the secondary structure, all the R groups will be facing outwards. The β sheet structure are quite common in nature and are favoured by the presence of aminoacids, glycine, alanine. • Silk and certain synthetic fibres such as nylon and orlon are composed of β-structure • Although the pleated sheets associated with structural proteins, it is also known to occur in 3-dimensinal structures of certain globular proteins, eg. The enzyme lysozyme and carboxypeptidase A.
  38. 38. The β- pleated sheet differs markedly from the rodlike α – helix: • The polypeptide chain in a β- pleated sheet, called a β-strand, has fully extended conformation, rather than being tightly coiled as in the α- helix. • The axial distance between adjacent amino acids in pleated sheets is 3.5 A˚, in the contrast with 1.5 A˚ for the α-helix. • β-Sheet is stabilized by hydrogen bonds between NH and CO groups in different polypeptide strands, where as, in α-helix, the hydrogen bonds are between NH and CO groups in the same strand. • In Alpha Helix -R groups of the amino acids are oriented outside of the helix while in Beta Pleated Sheet -R groups are directed to both inside and outside of the sheet. • Alpha helix prefers Ala, Leu, Met, Phe, Glu, Gln, His, Lys, Arg amino acids. Beta sheet prefers Tyr, Trp, (Phe, Met), Ile, Val, Thr, Cys.
  40. 40. PARALLEL β PLEATED SHEET. ANTIPARALLEL β PLEATED SHEET. Parallel – run in the same direction with longer looping sections between them (B) Anti-parallel – run in an opposite direction of its neighbor (A) If the N terminal ends of all the participating polypeptide chains lie on the same edge of the sheet, with C terminal ends on the opposite edge, the structure is known as parallel β pleated sheet. In contrast, if the direction of the chains alternates so that the alternating chains have their N terminal ends on the same side of the sheet, while their C terminal ends lie on the opposite edge, the structure is known as the antiparallel β pleated sheet.
  41. 41. • Both parallel and anti-parallel sheets have similar structures, although the repeat period is shorter (6.5 A˚ ) for the parallel conformation in comparison to anti-parallel conformation (7 A˚ )
  42. 42. Irregularities in regular structure • A β-bulge is a common noncompetitive irregularity found in antiparallel β-sheets. • It occurs between two normal β structure hydrogen bonds and involves two residues on one strand and one on other.
  43. 43. SUPER SECONDARY STRUCTURE OF PROTEINS • There are 4 types of super secondary structures in proteins. They are, • β-hairpin turn- two anti- parallel sheets linked by hairpin loop • β-meander- three anti- parallel sheets linked by hairpin loop • α-hairpin- two helices linked by hairpin loop • α-β-motif- two anti-parallel sheets linked by a helix
  44. 44. PROTEIN PACKING • occurs in the cytosol • involves interaction between secondary structure elements and solvent • yields tertiary structure
  45. 45. TERTIARY STRUCTURE • Tertiary structure refers to the three-dimensional structure of monomeric and multimeric protein molecules. • The α-helixes and β-pleated-sheets are folded into a compact globular structure. • It is a compact structure with hydrophobic side chains held interior while the hydrophillic groups are on the surface of the protein molecule. • Tertiary structure is stabilised by hydrogen bonds, disulfide bonds(-S-S), ionic interactions (electrostatic bonds),salt bridge and van der Waals force. • E.g. myoglobin, dihydrofolate reductase
  46. 46. PROTEIN INTERACTION • Occurs in the cytosol, in close proximity to other folded and packed proteins • involves interaction among tertiary structure elements of separate polymer chains
  47. 47. QUATERNARY STRUCTURE • Quaternary structure is the assembly of two or more polypeptide chains which are individually folded and compacted. Multimeric structure of different monomer. Proteins which possess quaternary structure are called as oligomers. • Each chain in the assembly is called as a subunit. • Commonly occuring examples are dimers, trimers, tetramers consisting of two, three and four polyprptide chains, respectively. A molecule, made up of a small number of subunits, is oligomer.
  48. 48. • The monomeric bonds are held together by non-covalent bonds namely hydrogen bonds, hydrophobic interactions and ionic bonds. • E.g. haemoglobin made up of four subunits, an allostreic protein.
  49. 49. • As a result of these covalent interactions, change occur in structure at one site of the protein molecule may cause drastic changes in properties at a distant site. Proteins exhibit this propery are called allosteric( protein with multiple ligand binding site). Not all multisubunit proteins exhibit allosteric effects, but many do. • Importance of oligomeric proteins- these protein play a significant role in the regulation of metabolism and cellular function.
  50. 50. DOMAINS • A domaindomain is a basic structural unit of a protein structure – distinct from those that make up the conformations • It is part of protein that can fold into a stable structure independently • Different domains can impart different functions to proteins • In larger proteins each domain is connected to other domains by short flexible regions of polypeptide. • Proteins can have one to many domains depending on protein size.
  51. 51. PROTEINS AT WORK • The conformation of a protein gives it a unique function • To work proteins must interact with other molecules, usually 1 or a few 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 • 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
  52. 52. LIGAND BINDING • The structure of a protein, for example, may change upon binding of its natural ligands, eg. a cofactor. In this a protein bound to the ligand is known as holo structure, of the unbound protein as apo structure.
  53. 53. FUNCTIONS OF PROTEIN • The total hereditary material of the cell or genotype dictates which type of protein the cell can produce. • STRUCTURAL PROTEINS - are fibrous and stringy and provide support. Eg. keratin, collagen, and elastin. Keratins strengthen protective coverings such as skin, hair, quills, feathers, horns, and beaks. Collagens and elastin provide support for connective tissues such as tendons and ligaments. • Proteins with catalytic activity( enzymes) are largely responsible for determining the phenotype or properties of cell. • DNA replication • Responding to stimuli
  54. 54. • ENZYMES - are referred to as catalysts that facilitate biochemical reactions. Eg. lactase and pepsin. Lactase breaks down the sugar lactose found in milk. Pepsin is a digestive enzyme that works in the stomach to break down proteins in food. • TRANSPORT PROTEINS - are carrier proteins which move molecules from one place to another around the body. Eg. hemoglobin and cytochromes. Hemoglobin transports oxygen through the blood via red blood cells. Cytochromes operate in the electron transport chain as electron carrier proteins. • CONTRACTILE PROTEINS - are responsible for movement. Eg. actin and myosin. These proteins are involved in muscle contraction and movement.
  55. 55. • HORMONAL PROTEINS - are messenger proteins which help to coordinate certain bodily activities. Eg. insulin, oxytocin. Insulin regulates glucose metabolism by controlling the blood-sugar concentration. Oxytocin stimulates contractions in females during childbirth. • ANTIBODIES - are specialized proteins involved in defending the body from antigens. One way antibodies counteract antigens is by immobilizing them so that they can be destroyed by white blood cells. • STORAGE PROTEINS - store amino acids. Eg. casein, ferritin. casein is a milk-based protein. Ferritin stores iron in hemoglobin.
  56. 56. LIST OF PROTEINS (AND PROTEIN COMPLEXES) • FIBROUS PROTEIN- 3-D structure is usually long and rod shaped Cytoskeletal proteins- keratin , myosin Extracellular matrix proteins- collagen, elastin • GLOBULAR PROTEIN- Compact shape like a ball with irregular surfaces. E.g. Enzymes are globular Plasma proteins- serum albumin Acute phase proteins- C-reactive protein Hemoproteins- Hemoglobin (oxyhemoglobin and deoxyhemoglobin) Transmembrane transport proteins- Glucose transporter (ion channel)
  57. 57. • Hormones and growth factors Growth factors - Epidermal growth factor (EGF) Fibroblast growth factor (FGF) Peptide hormones- Insulin, oxytoxin Steroid hormones- Estrogens, Progesterones • Receptors Transmembrane receptors- Rhodopsin Intracellular receptors- Estrogen receptor
  58. 58. • DNA-binding protein- Histones, Protamines • RNA-binding protein- SRRT (Serrate RNA effector molecule homolog) • Immune system proteins- Immunoglobins, Major histocompatibility antigens, T cell receptor • Nutrient storage/transport- Ferritin • Chaperone proteins- GroEL % • Enzymes
  59. 59. TOOLS • Blastp • CASTp • ClustalO • CyanoBase • CYS-REC • DIVEIN • Double prediction method • Expasy
  60. 60. • PDBsum tool • Protscale • Protparam • Putative phosphorylation site prediction • RAMPAGE • SAVES • SWISS-Model • VADAR
  61. 61. Thank you