CHAPTER 2 - PROTEIN STRUCTURE

F: THERMODYNAMICS AND IMFs IN PROTEIN STABILITY

BIOCHEMISTRY - DR. JAKUBOWSKI

Last Update:  3/2/16

F7.  Hydrophobic Effect and Protein Denaturation

The graph above shows a maximum in benzene insolubility.  As the temperature is decreased from that maximum,  benzene becomes more soluble in water.  Alternatively, as temperature rises to that temperature of maximal insolubility, the solubility of benzene decreases (just like nonpolar gases become increasingly insoluble with increasing temperature).    If you extrapolate the DG curve in this range of  decreasing temperature past the range shown on the graph, it would cross the X axis and become <0, implying benzene would be favored to dissolve in water.   Does the low temperature behavior of benzene/water interactions (becoming more soluble as the temperature is decreased from the maximum temperature for its insolubility) extend to and predict protein behavior at low temperature? (The following figures shows the analogy between benzene solubility in water and protein denaturation.

Figure:  Analogy between benzene solubility in water and protein denaturation

In the figure, F stands for a Phe side chains, which can be buried, sequestered from water as it would be in the native state of the protein, and exposed to water, as it might be in the denatured state.) The answer is yes, at low temperature. The analogy to benzene being more soluble at low temperature is the hydrophobic side chains in a protein becoming more likely to flip into water, denaturing the protein. The low temperature behavior would predict low temperature protein denaturation.  This phenomena has been observed.  Note that it doesn't require a change to a state when the nonpolar side chains prefer to be in water.  Just a change in that direction might be enough to tip the balance and lead to denaturation of the marginally stable protein. Please note that we are attempting to extrapolate the thermodynamic parameters associated with benzene solubility in water to the denaturation of a protein, NOT TO THE SOLUBILITY OF A PROTEIN IN WATER!

What about high temperature? Proteins denature as the temperature increases to the range that the DG curve for benzene reaches a peak. If the hydrophobic residues behave like benzene they would like to stay buried and not flip out into water as the temperature rises to the maximum in the DG curve.  Hence this predicts that the protein should become more stable.  What then explains the denaturation of proteins at high temperature? Another factor must account for it. What is it?

Remember the trans to gauche conformational changes in fatty acid residues in liposomes? As the temperature is increased, more conformations become available and occupied. Consider a protein.  At low temperature, their is only one native state and to pick a number, maybe 100 accessible denatured states. At high temperature, there is still only one native state, but possibly 1000 accessible denatured states.  More accurately, think of the protein existing in an ensemble of conformations.  As the temperature increases, more denatured states can be populated, compared to at lower temperatures, leading to an entropic driving force favoring unfolding.  Which way would the chain conformational entropy drive the protein at high temperature? Clearly, it would be driven to the most number of states - to the denatured state.  Hence a modern definition of the hydrophobic effect can explain low temperature denaturation, but not high temperature denaturation.

Remember when we discussed the thermodynamics of transfer of aliphatic alcohols from water to the pure alcohol? We decided that DGo was < 0 (favorable), and that DHo > 0 (disfavorable) and DSo > 0 (favorable). Also remember that these figures were derived at one temperature. We were somewhat surprised that DHo > 0 since this implies that from an enthalpic point of view, the alcohol-water interactions, or the water-water interactions surrounding the hydrocarbon chain are more favorable than the alcohol-alcohol interactions or bulk water-water interactions. The freeing of structured water surrounding the aliphatic chain when the alcohol is transferred to the pure alcohol is the driving force for the reaction. What happens at different temperatures? I hope it makes intuitive sense that the entropy effects will change with temperature, as described above. Likewise it makes sense that the enthalpy would change. Hence DH and DS for the transfer of amphiphiles into water will be a function of temperature - i.e. the reaction proceeds with a DCp.

Web Links:

• Hydrophobic Effect
• H-Bonds
• Hydrophobic Effect Demo

Online Literature:

K. A. Dill and J. L. MacCallum, The protein folding problem, 50 years on, Science 338, 1042-1046 (2012). (PDF) (Full Text Online) (podcast)

Southall, N.T., K.A. Dill, and A.D.J. Haymet.  A View of the Hydrophobic Effect.  Journal of Physical Chemistry B 106: 521-533 (2002). (PDF)

Summary of studies from small molecules (N-methyacetamide and benzene)

It is clear that proteins are not all that stable, and many contributions of varying magnitudes must sum to give the proteins marginal stability under physiological conditions. Hydrophobic interaction, defined in the new sense, must play a major role in stability. Also, since proteins are so highly packed compared to a lose denatured state, London Forces  must also play a significant part. (Remember dispersion forces are short range and become most significant under conditions of closest packing.) Opposing folding is the chain conformational entropy just described. Since proteins are so marginally stable, even one unpaired buried ionic side chain, or 1-2 unpaired buried H bond donors and acceptors   in the protein may be enough to "unravel" the native structure, leading to the denatured state.

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