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-bondsit 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 Hbond) leads to destabilization of mutant enzyme
Assumptions about changed hydrophobicity, etc
Specific H-Bonding Data
Quite a range of H-bond energiesvalid
approximation?
Hydrophobic Effect
Free energy of burying
nonpolar groups not
primarily vDWit 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.
