Thermodynamics of Protein Unfolding

1
Thermodynamics of Protein (Un)folding

• Free energy balance, energetics of protein
  structure, dominant forces in protein folding

Episode 1 Introduction, Thermal unfolding
http//www.biochem.oulu.fi/juffer/
2
Introduction (1)

• Folding is a thermodynamically driven process.
• Unique native structure represent the energetic
  balance of various types of interactions between
• Protein groups.
• Solvent (water, ions).
• Details of energetic balance has led to strong
  oscillations between different extreme opinions.

3
Introduction (2)

• Accepted point of view
• Hydrophobic interaction is the main driving force
  for folding.
• But if protein folding results from an overall
  increase in entropy, why do proteins denaturate
  at high temperature?

4
Introduction (3)

• Understanding of protein structure requires more
  than structural information only.
• Advance in understanding came by the acceptance
  of
• Calorimetric methods.
• Computational/theoretical methods.

5
Introduction (4)

• Calorimetric measurements have established that
• Proteins are thermodynamically macroscopic
  systems.
• Measured enthalpies, entropies and free energies
  are thermodynamic state functions.
• Heat capacity as a function of temperature leads
  directly to the partition function of
  macromolecules.
• Denaturated state may be regarded
  thermodynamically as the unfolded state
• interactions between protein groups are
  energetically insignificant.
• Free energy of folding is about equal to the free
  energy of denaturation.

6
Introduction (5)

• Role of solvent
• Essential
• Quantitative evaluation of hydration energies is
  difficult
• Depends on analysis of transfer free energies of
  small organic molecules to water.
• Is the contribution of different protein groups
  to the proteins total hydration energy additive?

7
Protein in a solvent
pH effects
8
Stability of protein structure

• Proteins are only marginally stable under the
  best of conditions.
• Protein structure can easily be disrupted by
  change in environmental conditions
• Temperature
• Variation in pH
• Increase of pressure
• Addition denaturants

Protein is denaturated
Denaturation is usually reversible
9
pH unfolding of staphylococcal nuclease A
Stability depends on pH
10
Temperature-induced unfolding of bovine
ribonuclease A
Thermal unfolding
11
Thermodynamic equilibrium
N native state U unfolded state
a average fraction of unfolding
Free energy of folding (f) the work that is
required to assemble the folded state.
12
Thermodynamics characteristics of proteins

• Denaturation experiments
• Reversible?
• Cooperative?
• Complete?
• Reliable?
• Temperature-induced unfolding is the best
  method

13
Thermal unfolding
Heat supplied to raise T
DCp? const?
Creighton, Proteins, structure and molecular
properties, Freeman, New York, 1993
14
Enthalpy and entropy of protein unfolding (1)
Gibbs free energy of unfolding (u) at temperature
T
At constant pressure
Heat capacity
15
Enthalpy and entropy of protein unfolding (2)
Assume DCp(T) ? Constant
TR is a reference temperature.
16
Enthalpy and entropy of protein unfolding (3)
With melting temperature Tm for TR
Generally referred to as the Modified
Gibbs-Helmholtz Equation
Experimental values for -RTlnKu as a function of
T can be fitted to obtain Tm, DHm and DCp.
Full characterisation of thermodynamics of
thermal unfolding
17
Enthalpy and entropy of protein unfolding (4)
Robertson and Murphy, Chemical Reviews, 97,
2151-1267, 1997
18
Enthalpy and entropy of protein unfolding (5)
19
Specific DCp(T) heat capacity increment

• It is always positive (larger for the unfolded
  state)
• Contains both effects due to configurational
  entropy and water accessibility (hydration).
• It is curved function having a slope that
  decreases with increasing T ? Not really
  constant!
• It correlates with surface area.

20
Heat capacity increment versus surface area
Nres (number of residues) correlates with surface
area.
Robertson and Murphy, Chemical Reviews, 97,
2151-1267, 1997
21
Free energy of unfolding
22
Enthalpy of unfolding
23
Entropy of unfolding
24
Heat capacity of unfolding
Not constant, likely to vanish above 1500C.
25
First conclusions

• The stability of very different proteins does not
  differ significantly 20-60 kJ/mol.
• This stability does not correlate with molecular
  weight.
• The stability is not great near physiological
  conditions
• Stability has a certain biological sense, perhaps
  important aspect of their evolution.