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Enzyme Kinetics & Protein Folding 9/7/2004

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Enzyme Kinetics & Protein Folding 9/7/2004

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  1. Enzyme Kinetics & Protein Folding9/7/2004

  2. Protein folding is “one of the great unsolved problems of science” Alan Fersht

  3. protein folding can be seen as a connection between the genome (sequence) and what the proteins actually do (their function).

  4. Protein folding problem • Prediction of three dimensional structure from its amino acid sequence • Translate “Linear” DNA Sequence data to spatial information

  5. 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

  6. 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.

  7. The sequence contains all the information to specify 3-D structure

  8. 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.

  9. Energy profiles during Protein Folding

  10. 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

  11. 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

  12. 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

  13. Protein denaturation • Can be denatured depending on chemical environment • Heat • Chemical denaturant • pH • High pressure

  14. 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

  15. 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

  16. The hydrophobic effect

  17. High ΔCp changes enthalpy significantly with temperature • For a two state reversible transition ΔHD-N(T2) = ΔHD-N(T1) + ΔCp(T2 – T1) • As ΔCp is positive the enthalpy becomes more positive • i.e. favors the native state

  18. High ΔCp changes entropy with temperature • For a two state reversible transition ΔSD-N(T2) = ΔSD-N(T1) + ΔCpT2 / T1 • As ΔCp is positive the entropy becomes more positive • i.e. favors the denatured state

  19. 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-N increases and causes the protein to unfold

  20. Cold unfolding • Due to the high value of ΔCp • Lowering the temperature lowers the enthalpy decreases Tc = T2m / (Tm + 2(ΔHD-N /ΔCp) i.e. Tm ~ 2 (ΔHD-N ) /ΔCp

  21. Measuring thermal denaturation

  22. Solvent denaturation • Guanidinium chloride (GdmCl) H2N+=C(NH2)2.Cl- • Urea H2NCONH2 • 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

  23. Solvent denaturation continued • Thus free energy is given by ΔGD-N = ΔGH2OD-N - mD-N [denaturant]

  24. 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

  25. 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

  26. Folding of a 20-mer poly Ala

  27. Unfolding of the DNA Binding Domain of HIV Integrase

  28. Two state transitions in multi-state reactions

  29. Rate determining steps

  30. Theories of protein folding • N-terminal folding • Hydrophobic collapse • The framework model • Directed folding • Proline cis-trans isomerisation • Nucleation condensation

  31. Molecular Chaperones • Three dimensional structure encoded in sequence • in vivo versus in vitro folding • Many obstacles to folding D<---->N  Ag

  32. 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

  33. 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

  34. GroEL

  35. 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

  36. GroEL is allosteric • Weak and tight binding states • Undergoes a series of conformation changes upon binding ligands • Hydrolysis of ATP follows classic sigmoidal kinetics

  37. Sigmoidal Kinetics • Positive cooperativity • Multiple binding sites

  38. Allosteric nature of GroEL

  39. GroEL changes affinity for denatured proteins • GroEL binds tightly • GroEL/GroES complex much more weakly

  40. 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

  41. 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

  42. Active site of GroEL • Residues 191-345 form a mini chaperone • Flexible hydrophobic patch

  43. Role of ATP hydrolysis

  44. The GroEL Cycle

  45. A real folding funnel

  46. 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

  47. 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 SAA1  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