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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
Amino Acids
Amino Acids
Polar: Hydrogen
bonding
Amino Acids
Acid/base:
Ion/ion
Primary Structure
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
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)
Factors in Folding/Unfolding
Stabilizing effects
Ionization/disulfide
bonds
Specific hydrogen
bonding
Hydrophobic effect
Destabilizing effects
Conformational entropy
Buried polar groups
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
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?
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.