Thermodynamics of Protein Folding Introduction and Literature Review

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

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

Amino Acids Nonpolar: vDW forces

Amino Acids Polar: Hydrogen bonding

Amino Acids Acid/base: Ion/ion

pH and Amino Acids

Primary Structure

Polar Peptide bonds

Secondary Structure: H-bonds

Secondary Structure: H-bonds

Tertiary Structure

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

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

Free Energy of Folding

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

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

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.

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

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

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

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

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

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?

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

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)

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

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

Summary: Contributions to RNase How valid are these approximations?

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?

Bibliography LiCata, V.K. et al. Proteins: Struct., Funct., Bioinf. 2004, 54, 616-621. LiCata, V.K. et al. Biochem. J. 2003, 374, 785-792. Pace, C.N., et al. FASEB J. 1996, 10, 75-83. Pace, C.N. Meth. Enz. 1995, 259, 538-554.