Lecture 18
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This document discusses various topics related to protein structure and folding. It provides details on methods for determining protein tertiary and quaternary structure using X-ray crystallography and NMR. It also discusses the protein folding problem, factors that influence folding, and diseases associated with misfolding. Molecular chaperones that assist with protein folding, such as GroEL, are described. Prions and amyloids, which form when proteins misfold, are also mentioned.
![Solvent denaturation continued
• Thus free energy is given by
ΔGD-N = ΔGH2O
D-N - mD-N[denaturant]](https://image.slidesharecdn.com/lecture18-141216084708-conversion-gate02/85/Lecture-18-28-320.jpg)
Lecture 18
- 1. Protein Structure Determination Protein Folding Molecular Chaperones Prions Alzyheimer’s
- 2. Tertiary Structure of Proteins Two methods: 1. X-RAY diffraction crystal structure 2. NMR solution structure This is a crystal X-ray diffraction pattern of sperm whale myoglobin
- 3. Electron density map 6 Å 2.0 Å 1.5 Å 1.1 Å From the diffraction pattern (spots and intensity) one can get a mathematical description of the electron density of a molecule. With proper model construction a 3-D image of the protein is constructed.
- 4. NMR
- 5. By using chemical shifts of backbone hydrogens and their chemical splitting bond angles can be determined. COSY NMR or Correlated Spectroscopy. By manipulating parameters protons that are close to each other in space but not linked through bonds can be determined by NOSY NMR or Nuclear Overhauser spectroscopy. Growing the protein in bacteria where the carbon source can be substituted by 13 C and the nitrogen by 15 N (stable isotope substitution) more restraints can be achieved. The liquid structure(s) can be determined as a group that fit a certain structure space.
- 6. Quaternary Structure and Symmetry Subunits can associate noncovalently, subunits are protomers if identical. Protomer subunits are symmetrically arranged Only rotational symmetry allowed. i.e. cyclic symmetry C2, C3, C6 etc. Dihedral symmetry N-fold intersects a two-fold rotational symmetry at right angles Other higher order types, octahedral or tetrahedral
- 8. Protein folding is “one of the great unsolved problems of science” Alan Fersht
- 9. protein folding can be seen as a connection between the genome (sequence) and what the proteins actually do (their function).
- 10. Protein folding problem • Prediction of three dimensional structure from its amino acid sequence • Translate “Linear” DNA Sequence data to spatial information
- 11. Why solve the folding problem? • Acquisition of sequence data relatively quick • Acquisition of experimental structural information slow • Limited to proteins that crystallize or stable in solution for NMR
- 12. Protein folding dynamics Electrostatics, hydrogen bonds and van der Waals forces hold a protein together. Hydrophobic effects force global protein conformation. Peptide chains can be cross-linked by disulfides, Zinc, heme or other liganding compounds. Zinc has a complete d orbital , one stable oxidation state and forms ligands with sulfur, nitrogen and oxygen. Proteins refold very rapidly and generally in only one stable conformation.
- 13. The sequence contains all the information to specify 3-D structure
- 14. Random search and the Levinthal paradox • The initial stages of folding must be nearly random, but if the entire process was a random search it would require too much time. Consider a 100 residue protein. If each residue is considered to have just 3 possible conformations the total number of conformations of the protein is 3100 . Conformational changes occur on a time scale of 10-13 seconds i.e. the time required to sample all possible conformations would be 3100 x 10-13 seconds which is about 1027 years. Even if a significant proportion of these conformations are sterically disallowed the folding time would still be astronomical. Proteins are known to fold on a time scale of seconds to minutes and hence energy barriers probably cause the protein to fold along a definite pathway.
- 15. Physical nature of protein folding • Denatured protein makes many interactions with the solvent water • During folding transition exchanges these non- covalent interactions with others it makes with itself
- 16. What happens if proteins don't fold correctly? • Diseases such as Alzheimer's disease, cystic fibrosis, Mad Cow disease, an inherited form of emphysema, and even many cancers are believed to result from protein misfolding
- 17. Protein folding is a balance of forces • Proteins are only marginally stable • Free energies of unfolding ~5-15 kcal/mol • The protein fold depends on the summation of all interaction energies between any two individual atoms in the native state • Also depends on interactions that individual atoms make with water in the denatured state
- 18. Protein denaturation • Can be denatured depending on chemical environment – Heat – Chemical denaturant – pH – High pressure
- 19. Thermodynamics of unfolding • Denatured state has a high configurational entropy S = k ln W Where W is the number of accessible states K is the Boltzmann constant • Native state confirmationally restricted • Loss of entropy balanced by a gain in enthalpy
- 20. Entropy and enthaply of water must be added • The contribution of water has two important consequences – Entropy of release of water upon folding – The specific heat of unfolding (ΔCp) • “icebergs” of solvent around exposed hydrophobics • Weakly structured regions in the denatured state
- 22. High ΔCp changes enthalpy significantly with temperature • For a two state reversible transition ΔHD-N(T2) = ΔHD-N(T1) + ΔCp(T2 – T1) • As ΔCpis positive the enthalpy becomes more positive • i.e. favors the native state
- 23. High ΔCp changes entropy with temperature • For a two state reversible transition ΔSD-N(T2) = ΔSD-N(T1) + ΔCpT2 / T1 • As ΔCpis positive the entropy becomes more positive • i.e. favors the denatured state
- 24. Free energy of unfolding • For ΔGD-N = ΔHD-N - TΔSD-N • Gives ΔGD-N(T2) = ΔHD-N(T1) + ΔCp(T2 – T1)- T2(ΔSD-N(T1) + ΔCpT2 / T1) • As temperature increases TΔSD-Nincreases and causes the protein to unfold
- 25. Cold unfolding • Due to the high value of ΔCp • Lowering the temperature lowers the enthalpy decreases Tc = T2 m / (Tm + 2(ΔHD-N/ΔCp) i.e. Tm ~ 2 (ΔHD-N) /ΔCp
- 27. Solvent denaturation • Guanidinium chloride (GdmCl) H2 N+ =C(NH2 )2 .Cl- • Urea H2 NCONH2 • Solublize all constitutive parts of a protein • Free energy transfer from water to denaturant solutions is linearly dependent on the concentration of the denaturant • Thus free energy is given by ΔGD-N = ΔHD-N - TΔSD-N
- 28. Solvent denaturation continued • Thus free energy is given by ΔGD-N = ΔGH2O D-N - mD-N[denaturant]
- 29. Acid - Base denaturation • Most protein’s denature at extremes of pH • Primarily due to perturbed pKa’s of buried groups • e.g. buried salt bridges
- 30. Two state transitions • Proteins have a folded (N) and unfolded (D) state • May have an intermediate state (I) • Many proteins undergo a simple two state transition D <—> N
- 31. Folding of a 20-mer poly Ala
- 32. Unfolding of the DNA Binding Domain of HIV Integrase
- 33. Two state transitions in multi-state reactions
- 36. Energy profiles during Protein Folding
- 38. Theories of protein folding • N-terminal folding • Hydrophobic collapse • The framework model • Directed folding • Proline cis-trans isomerisation • Nucleation condensation
- 39. Molecular Chaperones • Three dimensional structure encoded in sequence • in vivo versus in vitro folding • Many obstacles to folding D<---->N ↓ Ag
- 40. Molecular Chaperone Function • Disulfide isomerases • Peptidyl-prolyl isomerases (cyclophilin, FK506) • Bind the denatured state formed on ribozome • Heat shock proteins Hsp (DnaK) • Protein export & delivery SecB
- 41. What happens if proteins don't fold correctly? • Diseases such as Alzheimer's disease, cystic fibrosis, Mad Cow disease, an inherited form of emphysema, and even many cancers are believed to result from protein misfolding
- 42. GroEL
- 43. GroEL (HSP60 Cpn60) • Member of the Hsp60 class of chaperones • Essential for growth of E. Coli cells • Successful folding coupled in vivo to ATP hydrolysis • Some substrates work without ATP in vitro • 14 identical subunits each 57 kDa • Forms a cylinder • Binds GroES
- 44. GroEL is allosteric • Weak and tight binding states • Undergoes a series of conformation changes upon binding ligands • Hydrolysis of ATP follows classic sigmoidal kinetics
- 45. Sigmoidal Kinetics • Positive cooperativity • Multiple binding sites
- 46. Allosteric nature of GroEL
- 47. GroEL changes affinity for denatured proteins • GroEL binds tightly • GroEL/GroES complex much more weakly
- 48. GroEL has unfolding activity • Annealing mechanism • Every time the unfolded state reacts it partitions to give a proportion kfold/(kmisfold + Kfold) of correctly folded state • Successive rounds of annealing and refolding decrease the amount of misfolded product
- 49. GroEL slows down individual steps in folding • GroEL14 slows barnase refolding 400 X slower • GroEL14/GroES7 complex slows barnase refolding 4 fold • Truncation of hydrophobic sidechains leads to weaker binding and less retardation of folding
- 50. Active site of GroEL • Residues 191-345 form a mini chaperone • Flexible hydrophobic patch
- 51. Role of ATP hydrolysis
- 52. The GroEL Cycle
- 53. A real folding funnel
- 54. Amyloids • A last type of effect of misfolded protein • protein deposits in the cells as fibrils • A number of common diseases of old age, such as Alzheimer's disease fit into this category, and in some cases an inherited version occurs, which has enabled study of the defective protein
- 55. Known amyloidogenic peptides CJD spongiform encepalopathies prion protein fragments APP Alzheimer beta protein fragment 1-40/43 HRA hemodialysis-related amyloidosis beta-2 microglobin* PSA primary systmatic amyloidosis immunoglobulin light chain and fragments SAA 1 secondary systmatic amyloidosis serum amyloid A 78 residue fragment FAP I** familial amyloid polyneuropathy I transthyretin fragments, 50+ allels FAP III familial amyloid polyneuropathy III apolipoprotein A-1 fragments CAA cerebral amyloid angiopathy cystatin C minus 10 residues FHSA Finnish hereditary systemic amyloidosis gelsolin 71 aa fragment IAPP type II diabetes islet amyloid polypeptide fragment (amylin) ILA injection-localized amyloidosis insulin CAL medullary thyroid carcinoma calcitonin fragments ANF atrial amyloidosis atrial natriuretic factor NNSA non-neuropathic systemic amylodosis lysozyme and fragments HRA hereditary renal amyloidosis fibrinogen fragments
- 56. Transthyretin • transports thyroxin and retinol binding protein in the bloodstream and cerebrospinal fluid • senile systemic amyloidosis, which affects people over 80, transtherytin forms fibrillar deposits in the heart. which leads to congestive heart failure • Familial amyloid polyneuropathy (FAP) affects much younger people; causing protein deposits in the heart, and in many other tissues; deposits around nerves can lead to paralysis
- 57. Transthyretin structure • tetrameric. Each monomer has two 4-stranded β-sheets, and a short α-helix. Anti- parallel beta-sheet interactions link monomers into dimers and a short loop from each monomer forms the main dimer-dimer interaction. These pairs of loops keep the two halves of the structure apart forming an internal channel.
- 58. Fibril structure • Study of the fibrils is difficult because of its insolubility making NMR solution studies impossible and they do not make good crystals • X-ray diffraction, indicates a pattern consistent with a long β-helical structure, with 24 β-strands per turn of the β-helix.
- 59. Formation of proto-filaments • Four twisted β-helices make up a proto-filament (50-60A) • Four of these associate to form a fibril as seen in electron microscopy (130A)
Editor's Notes
- #21: (ΔCp) specific heat is defined as the energy to raise one mole through one degree (Celsius)
- #22: (ΔCp) specific heat is defined as the energy to raise one mole through one degree (Celsius)
- #23: (ΔCp) specific heat is defined as the energy to raise one mole through one degree (Celsius)
- #24: (ΔCp) specific heat is defined as the energy to raise one mole through one degree (Celsius)
- #25: (ΔCp) specific heat is defined as the energy to raise one mole through one degree (Celsius)
- #26: (ΔCp) specific heat is defined as the energy to raise one mole through one degree (Celsius)
- #28: (ΔCp) specific heat is defined as the energy to raise one mole through one degree (Celsius)
- #29: (ΔCp) specific heat is defined as the energy to raise one mole through one degree (Celsius)
- #30: (ΔCp) specific heat is defined as the energy to raise one mole through one degree (Celsius)
- #31: (ΔCp) specific heat is defined as the energy to raise one mole through one degree (Celsius)
- #34: (ΔCp) specific heat is defined as the energy to raise one mole through one degree (Celsius)
- #43: GroES is the cap for GroEL Ask question of human homologs of GroEL & examples of chaperones found from a sgx program
- #44: GroES is the cap for GroEL Ask question of human homologs of GroEL & examples of chaperones found from a sgx program
- #46: sThe activated (blue) curve is ~hyperbolic. In the presence of activator, the enzyme appears to be in the R-form. In the absence of the activator or the presence of inhibitor (black and red curves) appear to have decreasing R-form characteristics and more the curve of the T-form of the allosteric enzyme.
- #47: sThe activated (blue) curve is ~hyperbolic. In the presence of activator, the enzyme appears to be in the R-form. In the absence of the activator or the presence of inhibitor (black and red curves) appear to have decreasing R-form characteristics and more the curve of the T-form of the allosteric enzyme.
- #51: Notice it forms a single catalytic domain
- #53: Notice it forms a single catalytic domain
- #55: Notice it forms a single catalytic domain
- #56: Notice it forms a single catalytic domain
- #57: Notice it forms a single catalytic domain
- #58: Notice it forms a single catalytic domain
- #59: Notice it forms a single catalytic domain
- #60: Notice it forms a single catalytic domain