Thermodynamics of Protein Folding Introduction and Literature Review.
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Transcript of Thermodynamics of Protein Folding Introduction and Literature Review.
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.