Thermodynamics of Protein Folding

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Transcript Thermodynamics of Protein Folding

Thermodynamics of Protein
Introduction and Literature Review
• 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
Amino Acids
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
– Specific hydrogen
– Hydrophobic effect
• Destabilizing effects
– Conformational entropy
– Buried polar groups
Specific Hydrogen Bonding
• Folding not only forms H-bonds—it also destroys
• 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 Hbond) leads to destabilization of mutant enzyme
– Assumptions about changed hydrophobicity, etc
Specific H-Bonding Data
• Quite a range of H-bond energies—valid
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
• 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
• 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
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?
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.