1. Thermodynamics of Protein Folding
Introduction and Literature Review

2. Overview Applications of what we have learned Intermolecular forces
Effect of acid/base chemistry
Calorimetry
Free energy of folding
Equilibrium and stability of solvation
Entropy: The hydrophobic effect

3. Protein Folding Activity of proteins depends on 3-D shape
Primary structure
Secondary and Tertiary structure

5. Amino Acids
Nonpolar: vDW forces

6. Amino Acids
Polar: Hydrogen bonding

7. Amino Acids
Acid/base:
Ion/ion

8. pH and Amino Acids

9. Primary Structure

10. Polar Peptide bonds

11. Secondary Structure: H-bonds

12. Secondary Structure: H-bonds

13. Tertiary Structure

14. Thermodynamics of Taq Work from LiCata, et al. Polymerase
E. coli
Thermus aquaticaus (Taq)
Active fragments
Klenow
Klentaq

15. Calorimetry of Taq
Differential Scanning Calorimetry measures difference in energy needed to keep sample and reference increasing in temperature
Marks energy input into non-kinetic mode (degree of freedom)
DH = CDT

16. Free Energy of Folding

17. Free Energy of Folding for Taq
Experiment
pH 9.5
Guanidinium chloride
To compare, need same conditions for both without aggregation of proteins
Taq DGunfold = 27 kcal/mol
Klenow DGunfold = 4.5 kcal/mol

18. Structural Basis of Taq Stability
Steitz et al. suggest Taq has 4 additional internal H-bonds and 2 additional ion/ion interactions compared to Klenow
Waksman et al. suggest fewer unfavorable electrostatic charges lead to global rearrangement of electrostatic distribution and more buried nonpolar space
LiCata suggests that unfolded Taq has more surface area, leading to greater relative destabilization of unfolded relative to folded

19. Thermodynamic Principles of Protein Folding
Very difficult to determine how all factors blend together to give overall DGfolding
Use of averages contributions, but
Each protein is unique
Large stabilization factors, large destabilization factors, but small difference between them
Use RNase T1 as a model for study (because structure is well known and many mutants have been studied)
Based on work of Pace, et al.

20. Factors in Folding/Unfolding
Stabilizing effects
Ionization/disulfide bonds
Specific hydrogen bonding
Hydrophobic effect
Destabilizing effects
Conformational entropy
Buried polar groups

21. Specific Hydrogen Bonding
Folding not only forms H-bonds—it also destroys them!
But which are stronger?
Transient solvent H-bonds
Specific H-bonds
Mutants show that formation of specific H-bonds stabilize protein by average of 1.6 kcal
Replacing asparagine H-bond with alanine (no H-bond) leads to destabilization of mutant enzyme
Assumptions about changed hydrophobicity, etc

22. Specific H-Bonding Data
Quite a range of H-bond energies—valid approximation?

23. Hydrophobic Effect
Free energy of burying nonpolar groups not primarily vDW—it is an entropic effect
Water “freezes” around nonpolar surface—clatherate shell
vDW important—cavities are destabilizing
Traditionally, thought to be actual driving force of protein folding

24. Hydrophobic Effect: Quantitative
Free energy of transfer between water and octanol—transfer of side chain from water to model of non-polar protein core
Data suggest about 0.8 kcal stabilization for each –CH2 group buried
Mutant models show energy difference of 1.1 kcal/methylene
Suggests that burial of hydrophobic group has van der Waals contribution

25. Conformational Entropy
Spolar and Record used calorimetry to predict an average entropy of folding of -5.6 e.u.
What does this translate to for the free energy change for freezing conformational entropy in RNase T1 (104 residues) at 25 oC?

26. Burying Polar Groups
Water dielectric constant vs protein dielectric constant
Even if H-bonding is maintained, it is unfavorable to put polar group in nonpolar environment
Model: Partitioning of amino acid sidechains and peptide bonds between water and octanol
Determine K
Calculate DG

27. Burying Polar Groups
DG of transfer between water and octanol is thought to be best model (Transfer between water and cyclohexane also includes loss of H-bond)

28. Summary: Contributions to RNase
Conformational entropy: calculated
Peptide buried = 73.4 peptides (1.1 kcal/peptide)
Polar buried based on previous table

29. Summary: Contributions to RNase
Ionization and disulfide: experimental
Hydrophobic groups: from DGtr
H-bonding = 1.6 kcal (104 H-bonds)

30. Summary: Contributions to RNase
How valid are these approximations?

31. Conclusions: Hydrophobic Effect or H-Bonding?
Pace is making the case for the importance of H-bonds vs hydrophobic effect in protein folding. How did he do?

32. Bibliography
LiCata, V.K. et al. Proteins: Struct., Funct., Bioinf. 2004, 54, LiCata, V.K. et al. Biochem. J. 2003, 374, Pace, C.N., et al. FASEB J. 1996, 10, Pace, C.N. Meth. Enz. 1995, 259,