thermodynamics of protein folding introduction and literature review
TRANSCRIPT
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Thermodynamics of Protein Folding
Introduction and Literature Review
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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
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Protein Folding
• Activity of proteins depends on 3-D shape• Primary structure• Secondary and Tertiary structure
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Amino Acids
• Nonpolar: vDW forces
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Amino Acids
• Polar: Hydrogen bonding
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Amino Acids
Acid/base:Ion/ion
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pH and Amino Acids
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Primary Structure
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Polar Peptide bonds
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Secondary Structure: H-bonds
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Secondary Structure: H-bonds
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Tertiary Structure
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Thermodynamics of Taq
• Work from LiCata, et al.
• Polymerase– E. coli– Thermus
aquaticaus (Taq)• Active fragments– Klenow– Klentaq
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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
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Free Energy of Folding
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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
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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
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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.
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Factors in Folding/Unfolding
• Stabilizing effects– Ionization/disulfide
bonds– Specific hydrogen
bonding– Hydrophobic effect
• Destabilizing effects– Conformational entropy– Buried polar groups
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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
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Specific H-Bonding Data
• Quite a range of H-bond energies—valid approximation?
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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
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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
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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?
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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
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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)
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Summary: Contributions to RNase
• Conformational entropy: calculated• Peptide buried = 73.4 peptides (1.1 kcal/peptide)• Polar buried based on previous table
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Summary: Contributions to RNase
• Ionization and disulfide: experimental• Hydrophobic groups: from DGtr
• H-bonding = 1.6 kcal (104 H-bonds)
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Summary: Contributions to RNase
How valid are these approximations?
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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?
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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.