Protein structure Lecture for M Sc biology students
- 1. Protein Structure Dr. Anuj Kumar Scientist Indian Council of medical research
- 2. Fibrous proteins have a structural role 2 Source:http://www.prideofindia.net/images/nails.jpg http://opbs.okstate.edu/~petracek/2002%20protein%20structure%20function/CH06/Fig%2006-12.GIF http://my.webmd.com/hw/health_guide_atoz/zm2662.asp?printing=true Keratin •Tough insoluble protein •Makes up the quills of echidna, your hair and nails and the rattle of a rattle snake. •Structure comes from α-helices that are cross- linked by disulfide bonds. Collagen •Collagen most abundant protein in vertebrates. •Collagen fibers are a major portion of tendons, bone and skin. •α-helices of collagen make up a Triple helix structure giving it tough and flexible properties. Fibroin fibers •Makes the silk spun by spiders and silk worms stronger weight for weight than steel! •The soft and flexible properties come from the β- structure.
- 4. Hierarchical nature of protein structure Primary structure (Amino acid sequence) ↓ 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
- 5. Protein Structure: Conformation • The spatial arrangement of atoms in a protein or any part of protein in called conformation (shape/structure) • Conformation stabilized by weak interactions and disulphide bond – Difference between folded and unfolded approx 20-65 kJ/mol – Weak interactions 0.4-30 kJ/mol (Peptide bond 200-460 kJ/mol) • Proteins in any of their functional , folded conformations are called native proteins • In general, native conformation is lowest ΔG
- 6. Weak interactions 1. Hydrophobic effects – Oil droplet in water, solvation layer – Inner core of protein consists of hydrophobic amino acids 1. Ion pair/salt bridges – Increase with decrease in dielectric constant (ε) (example change from water (ε=80) to n-hexane (ε = 1.89) 1. H-bonds – Length approx 1.5-2.5 Å 1. van der Waals interaction – Dipole-dipole interactions – Work in distance of 0.3 to 0.6nm
- 7. Hydrogen Bonding • Involves three atoms: – Donor electronegative atom (D) (Nitrogen or Oxygen in proteins) – Hydrogen bound to donor (H) may be from –N atom –NH2 – Acceptor electronegative atom (A) in close proximity may be Oxygen atom Dδ- – Hδ+ Aδ-
- 8. D-H Interaction • Polarization due to electron withdrawal from the hydrogen to D giving D partial negative charge and the H a partial positive charge e.g. H2O • Proximity of the Acceptor A causes further charge separation D – H A δ- δ+ δ-
- 9. D-H Interaction • Polarization due to electron withdrawal from the hydrogen to D giving D partial negative charge and the H a partial positive charge • Proximity of the Acceptor A causes further charge separation • Result: – Closer approach of A to H – Higher interaction energy than a simple van der Waals interaction D – H A δ- δ+ δ- One liter Water=55.5M=55.5x 6.02x1023 =334.11x1023 33,411,000,000,000,000,000,000,000 water molecules per liter!
- 10. Hydrogen Bonding And Secondary Structure alpha-helix beta-sheet
- 13. X-Ray Crystallography • Protein is crystallized • Bombard with X-rays, record scattering diffraction patterns • Determine electron density map from scattering and phase via Fourier transform. • Use electron density and biochemical knowledge of the protein to refine and determine a model
- 14. An X-ray diffraction photograph of a crystal of sperm whale myoglobin. The intensity of each diffraction maximum (the darkness of each spot) is a function of the crystal’s electron density. The distance between the spots is function of atomic distance Bragg try to solve the structure of α-helix X-ray diffraction photograph
- 15. Peptide Bond – Rigid and Planar • A resonance in peptide bond (40% double bond character) • Partial negative charge on Oxygen and positive charge on Nitrogen – Dipole • All six atoms of the peptides are on single plane • Three dihedral angle / torsion angle – Φ angle between C-N-Cα -C – ψ angle between N-Cα-C-N – ω angle between Cα-N-C-Cα • N-C (ω) cannot rotate freely • Rotation allowed b/w N-Cα (Φ) and Cα-C(ψ)
- 16. Folding of the backbone chain: Phi (Φ)and Psi (ψ) Angles Degrees of Freedom = 2
- 17. Ramachandran plot • Phi (Φ) and Psi (ψ) rotate, allowing the polypeptide to assume its various conformations • Some conformations of the polypeptide backbone result in steric hindrance and are disallowed • Glycine has no side chain and is therefore conformationally highly flexible (it is often found in turns) • Dr. Ramachandran used coordinated of known di-, tri- and polypeptide of that time including collagen and silk fibroin Phi (Φ) Psi (ψ)
- 18. Ramachandran plot What type of Ramchandran plot for poly Glycine and poly Proline?
- 19. Ramachandran plot no steric clashes permitted if atoms are more closely spaced
- 20. Secondary structure α-helix β-sheet Secondary structures, α-helix and β- sheet, have regular hydrogen-bonding patterns. Well defined and fixed pattern of H-bonding Φ angle and ψ angle remain same
- 21. While working on a fibrous protein α-keratin, Pouling and Corey determined α-Helix L-amino acids favors right-handed alpha helix. Individual dipole of peptide taken together results in a helix dipole 1.5 Å increase in height for 180° rotation 43 63 −= −= ψ φ α Helix 3.6 amino acids/turn
- 22. Constraints affecting stability of helix • The intrinsic propensity of aa – V, T, I destabilize due to steric clashes – S, D, N destabilize due to H-donor – P destabilize due to fix phi angle (60 degrees) and No H atom to donate for H-bonding – Glycine highly flexible • Aa at the end
- 23. Pleated appearance of Beta sheet. β strand • Extended conformation, zigzag structure • Adjacent aa distance 3.5 A • R groups in opposite direction • Parallel and anti parallel • Interstrand H-bonds – in-line in antiparallel strands – distorted in parallel strands
- 25. β Turns • Four residues • H-bonding between i and i+3 • Gly and Pro often occur in β turns
- 26. Motif or folds • Are not independently stable • Super secondary structures • Recongnizable folding patterns involving two or more secondary structures • Basis of structure classification Domains • Are independently stable • Can have individual function
- 27. Tertiary structure Fold defines function 3D structure of HA protein of Influenza A/H1N1; Known epitopes highlighted in different colors Visualization: DiscoveryStudio Tertiary Structure
- 28. Myoglobin First protein in atomic structure L,V, M and F in the interior Very compact with very less space Only H in the inside core
- 29. Quaternary Structure • non-linear • 3 dimensional • global, and across distinct amino acid polymers • formed by hydrogen bonding, covalent bonding, hydrophobic packing and hydrophilic exposure • favorable, functional structures occur frequently and have been categorized
- 30. Multimeric Proteins Macromolecular Assemblies Hemoglobin: A tetramer Quaternary Structure: RuBisCO protein : L8S8
- 33. SDS PAGE and Quaternary Structure
- 34. Intrinsically Disordered Proteins • Lack of hydrophobic core • Large number of charged amino acids in core • A third of all human proteins are intrinsically disordered • Large number of viral proteins are intrinsically disordered • Intrinsically Disordered Proteins are at the centre of protein- protein interaction network
- 35. Protein denaturation and folding Christian Anfinsen experiment • Denaturation by temperature, pH, chaotropic agent • Ribonuclease A denaturation • By urea and reducing agent • After removal of urea and reducing agent complete gain in activity • Primary sequence have all the information required to gain native structure
- 36. Protein folding • Levinthal’s paradox – 100aa protein on random folding – 10-13 sec/conformation – 1077 years • Protein folding not random process • But a stepwise process
- 37. Homology modeling and threading • Homology modeling = knowledge-based approach, given a sequence database, use multiple sequence alignment on this database to identify structurally conserved regions and construct structure backbone and loops based on these regions, restore side-chains and refine through energy minimization (apply to proteins that have high sequence similarity to those in the database) • Threading = knowledge-based approach, given a structure database of interest (e.g. one that provides a limited set of possible structures per given sequence for fold recognition, one that provides a one structure per given limited set of possible sequences for inverse folding) use scoring functions and correlations from this database to derive structure that is in agreement (apply to proteins with moderate sequence similarity to those in the database)
- 38. Red Blood Cell (Erythrocyte)
- 39. Hemoglobin: Background • Protein in red blood cells • Composed of four subunits, each containing a heme group: a ring-like structure with a central iron atom that binds oxygen • Picks up oxygen in lungs, releases it in peripheral tissues (e.g. muscles)
- 40. • Sickle-cell anemia resulting from one residue change in hemoglobin protein • Replace highly polar (hydrophilic) glutamate with nonpolar (hydrophobic) valine
- 41. Sickle-cell mutation in hemoglobin sequence
- 42. Normal Trait • Hemoglobin molecules exist as single, isolated units in RBC, whether oxygen bound or not • Cells maintain basic disc shape, whether transporting oxygen or not
- 43. Sickle-cell Trait • Oxy-hemoglobin is isolated, but de- oxyhemoglobin sticks together in polymers, distorting RBC • Some cells take on “sickle” shape
- 44. RBC Distortion • Hydrophobic valine replaces hydrophilic glutamate • Causes hemoglobin molecules to repel water and be attracted to one another • Leads to the formation of long hemoglobin filaments • Filaments distort the shape of red blood cells (analogy: icicle in a water balloon) • Rigid structure of sickle cells blocks capillaries and prevents red blood cells from delivering oxygen
- 45. Sickle-cell Trait • Oxy-hemoglobin is isolated, but de- oxyhemoglobin sticks together in polymers, distorting RBC • Some cells take on “sickle” shape • When hemoglobin again binds oxygen, again becomes isolated • Cyclic alteration damages hemoglobin and ultimately RBC itself
- 46. Sickle-cell
- 48. Thank you