Protein Structure Determination Protein Folding Molecular Chaperones Prions Alzyheimer’s

1. Protein Structure DeterminationProtein FoldingMolecular ChaperonesPrionsAlzyheimer’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 13C and the nitrogen by 15N (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

21. The hydrophobic effect

22. 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

23. 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

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-N increases and causes the protein to unfold

25. 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

26. Measuring thermal denaturation

27. 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

28. Solvent denaturation continued • Thus free energy is given by ΔGD-N = ΔGH2OD-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

34. Rate determining steps

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