1. Enzyme Kinetics & Protein Folding 9/7/2004

2. “one of the great unsolved problems of science”
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 Conformational changes occur on a time scale of seconds i.e. the time required to sample all possible conformations would be 3100 x 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

11. 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

12. 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

13. 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

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

15. 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

16. 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
(ΔCp) specific heat is defined as the energy to raise one mole through one degree (Celsius)

17. The hydrophobic effect
(ΔCp) specific heat is defined as the energy to raise one mole through one degree (Celsius)

18. 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
(ΔCp) specific heat is defined as the energy to raise one mole through one degree (Celsius)

19. 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
(ΔCp) specific heat is defined as the energy to raise one mole through one degree (Celsius)

20. 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
(ΔCp) specific heat is defined as the energy to raise one mole through one degree (Celsius)

21. 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
(ΔCp) specific heat is defined as the energy to raise one mole through one degree (Celsius)

22. Measuring thermal denaturation

23. 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
(ΔCp) specific heat is defined as the energy to raise one mole through one degree (Celsius)

24. Solvent denaturation continued
Thus free energy is given by
ΔGD-N = ΔGH2OD-N - mD-N [denaturant]
(ΔCp) specific heat is defined as the energy to raise one mole through one degree (Celsius)

25. 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
(ΔCp) specific heat is defined as the energy to raise one mole through one degree (Celsius)

26. Two state transitions D <—> N
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
(ΔCp) specific heat is defined as the energy to raise one mole through one degree (Celsius)

27. Folding of a 20-mer poly Ala

28. Unfolding of the DNA Binding Domain of HIV Integrase

29. Two state transitions in multi-state reactions
(ΔCp) specific heat is defined as the energy to raise one mole through one degree (Celsius)

30. Rate determining steps

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

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

35. 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

36. 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

37. GroEL
GroES is the cap for GroEL Ask question of human homologs of GroEL & examples of chaperones found from a sgx program

38. 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
GroES is the cap for GroEL Ask question of human homologs of GroEL & examples of chaperones found from a sgx program

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

40. Sigmoidal Kinetics Positive cooperativity Multiple binding sites
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.

41. Allosteric nature of GroEL
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.

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

43. 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

44. 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

45. Active site of GroEL Residues 191-345 form a mini chaperone
Flexible hydrophobic patch
Notice it forms a single catalytic domain

46. Role of ATP hydrolysis

47. The GroEL Cycle
Notice it forms a single catalytic domain

48. A real folding funnel

49. 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
Notice it forms a single catalytic domain

50. 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
Notice it forms a single catalytic domain

51. 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
Notice it forms a single catalytic domain

52. Transthyretin structure
tetrameric. Each monomer has two 4-stranded b-sheets, and a short a-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.
Notice it forms a single catalytic domain

53. 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 b-helical structure, with 24 b-strands per turn of the b-helix.
Notice it forms a single catalytic domain

54. Formation of proto-filaments
Four twisted b-helices make up a proto-filament (50-60A)
Four of these associate to form a fibril as seen in electron microscopy (130A)
Notice it forms a single catalytic domain