14Thermodynamics of Taq Work from LiCata, et al. Polymerase E. coliThermus aquaticaus (Taq)Active fragmentsKlenowKlentaq
15Calorimetry of TaqDifferential Scanning Calorimetry measures difference in energy needed to keep sample and reference increasing in temperatureMarks energy input into non-kinetic mode (degree of freedom)DH = CDT
17Free Energy of Folding for Taq ExperimentpH 9.5Guanidinium chlorideTo compare, need same conditions for both without aggregation of proteinsTaq DGunfold = 27 kcal/molKlenow DGunfold = 4.5 kcal/mol
18Structural Basis of Taq Stability Steitz et al. suggest Taq has 4 additional internal H-bonds and 2 additional ion/ion interactions compared to KlenowWaksman et al. suggest fewer unfavorable electrostatic charges lead to global rearrangement of electrostatic distribution and more buried nonpolar spaceLiCata suggests that unfolded Taq has more surface area, leading to greater relative destabilization of unfolded relative to folded
19Thermodynamic Principles of Protein Folding Very difficult to determine how all factors blend together to give overall DGfoldingUse of averages contributions, butEach protein is uniqueLarge stabilization factors, large destabilization factors, but small difference between themUse 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.
20Factors in Folding/Unfolding Stabilizing effectsIonization/disulfide bondsSpecific hydrogen bondingHydrophobic effectDestabilizing effectsConformational entropyBuried polar groups
21Specific Hydrogen Bonding Folding not only forms H-bonds—it also destroys them!But which are stronger?Transient solvent H-bondsSpecific H-bondsMutants show that formation of specific H-bonds stabilize protein by average of 1.6 kcalReplacing asparagine H-bond with alanine (no H-bond) leads to destabilization of mutant enzymeAssumptions about changed hydrophobicity, etc
22Specific H-Bonding Data Quite a range of H-bond energies—valid approximation?
23Hydrophobic EffectFree energy of burying nonpolar groups not primarily vDW—it is an entropic effectWater “freezes” around nonpolar surface—clatherate shellvDW important—cavities are destabilizingTraditionally, thought to be actual driving force of protein folding
24Hydrophobic Effect: Quantitative Free energy of transfer between water and octanol—transfer of side chain from water to model of non-polar protein coreData suggest about 0.8 kcal stabilization for each –CH2 group buriedMutant models show energy difference of 1.1 kcal/methyleneSuggests that burial of hydrophobic group has van der Waals contribution
25Conformational 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?
26Burying Polar GroupsWater dielectric constant vs protein dielectric constantEven if H-bonding is maintained, it is unfavorable to put polar group in nonpolar environmentModel: Partitioning of amino acid sidechains and peptide bonds between water and octanolDetermine KCalculate DG
27Burying Polar GroupsDG of transfer between water and octanol is thought to be best model (Transfer between water and cyclohexane also includes loss of H-bond)
28Summary: Contributions to RNase Conformational entropy: calculatedPeptide buried = 73.4 peptides (1.1 kcal/peptide)Polar buried based on previous table
29Summary: Contributions to RNase Ionization and disulfide: experimentalHydrophobic groups: from DGtrH-bonding = 1.6 kcal (104 H-bonds)
30Summary: Contributions to RNase How valid are these approximations?
31Conclusions: 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?
32BibliographyLiCata, 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,