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