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Thermodynamics of Protein Folding Introduction and Literature Review.

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Presentation on theme: "Thermodynamics of Protein Folding Introduction and Literature Review."— Presentation transcript:

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


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)  H = C  T

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  G unfold = 27 kcal/mol Klenow  G unfold = 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  G folding – 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 –CH 2 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 o C?

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  G

27 Burying Polar Groups  G 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 DG tr 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,

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