1. The Hydrophobic Effect. Hydrophobic Interactions: These are very important because the main driving force for protein folding is minimization of the solvent-exposed non-polar (hydrophobic) surface area. This decreases about 3- 4-fold on folding. One general observation in protein and or membrane structure is the fact that non-polar residues sequester away from an aqueous environment. This fact is not surprising. The explanation for this fact is incomplete. Some ideas are presented below.

2. Consider a simple hydrocarbon (propane) C 3 H 8 introduced into water (i.e. transferred from pure liquid). C 3 H 8 (l)  C 3 H 8 (aq)  H o 298 = -8kJ/mole (favorable !!)  S o 298 = -80J/ o K-mole (unfavorable)  G o 298 = +16kJ/mole (unfavorable) The formation of oil drops is an entropy-driven process. The question is “Why ?”.

4. There are numerous scales of amino acid hydrophobicity. Typically the hydrophobicity is measured in terms of the free energy of transfer (  G tr ) of the group of interest from aqueous solution to a non-polar solvent, often octanol. In general, a good correlation is found between  G tr values and other measures of hydrophobicity, as well as the accessible surface area of the amino acid side chains.

7. Unlabeled dots are for various hydrocarbons. The line extrapolates back to the origin and has a slope of 24 cal/  2. Labeled dots refer to the side chains of the amino acids. The line passing through the ala,val, phe and leu has a slope of 22 cal/  2. The other amino acids have polar groups and consequently lower hydrophobicities than those expected from their surface areas. Correlation: hydrophobicity with accessible surface area. 37

8. The formation of oil drops is an entropy-driven process. The question is “Why ?”. (1) Old explanation Each hydrocarbon molecule introduced into water disrupts its H-bonding network. The hydrocarbons do not interact with H 2 O strongly. Water molecules around the hydrocarbon orient themselves in such a way which reforms the H-bonds that were disrupted by the hydrocarbon. The net effect is that water molecules around the hydrocarbon are more ordered compared to pure water. This gives rise to  S<0. There is little change in the number of H bonds so H is small. The magnitude of this effect is related to the area occupied by the hydrocarbon. The coalescence of hydrocarbons reduces the area on which ordered water can form. ( NOTE: There is no such thing as a hydrophobic bond. The interaction is the result of the combined effects of London, van der Waals, and dispersion forces).

9. (2) NEW, revised explanation The nature of the hydrophobic effect has been the subject of endless controversy since Kauzman's seminal contribution in 1959 (Adv. Prot. Chem. 14, 1-63, 1959). It is reasonably clear that the hydrophobic effect is a consequence of the special properties of liquid water, most probably a combination of the strong hydrogen bonding and the small size of water. It is now reasonable to suggest that the hydrophobic effect is not just an entropic effect as was postulated (see above !!) for many years, but has both entropic and enthalpic contributions which vary dramatically with temperature. Thus at room temperature the effect happens to be mainly entropic. The underlying basis of the hydrophobic interaction is the lack of strong favorable interactions between polar water molecules and non-polar molecules. This effectively leads to an increase in the interaction between the non-polar molecules. A simple concept for understanding the effect is to consider it necessary to create a cavity in the solvent water in order to place a non-polar molecule in it. Thus there is a local increase in the structure and order of the water (entropy) and also increased number of H-bonds (enthalpy). As you know, water can form a maximum of 4 H-bonds per molecule, but as found in normal liquid water has an average of around 3.

10. The enthalpy contribution of the hydrophobic interaction is approx. 0 around 20°C, i. e. room temperature, whereas the entropy contribution becomes 0 around 140°C. At temperatures much above room temperature there is increasingly less ordering of the water molecules around a non- polar group. As the temperature decreases the strength of the hydrophobic interaction decreases: this is the opposite effect to that of H- bonds, which become stronger at lower temperatures. There are numerous scales of amino acid hydrophobicity. Typically the hydrophobicity is measured in terms of the free energy of transfer (  G tr ) of the group of interest from aqueous solution to a non-polar solvent, often octanol. In general, a good correlation is found between  G tr values and other measures of hydrophobicity, as well as the accessible surface area of the amino acid side chains.

11. Non-covalent Forces in Proteins 1.Hydrogen bonds 2.Salt-bridges 3.Dipole-dipole interactions 4.Van der Waals forces 5.Hydrophobic effect A typical protein would contain a few salt-bridges, several hundred hydrogen bonds and several thousand van der Waals interactions. In spite of all these interactions... Proteins are only marginally stable Typical  G values for folding of proteins are in the range of -5 to -15 kcal/mol i.e. not much greater than the energy of 2 or 3 hydrogen bonds. This is because of several effects which cancel each other out.

12. The enthalpy change of protein folding (  H) is dominated by hydrogen bonds. In the unfolded state the polar groups of the protein will H-bond to solvent molecules and in the folded state these polar groups will H- bond with each other. Hence the overall enthalpy change on folding is small. The hydrophobic effect is thought to make the largest contribution to  G. The hydrophobic effect attributes the poor solubility of non-polar groups in water to the ordering of the surrounding water molecules causing them to form an ice-like cluster (see Figure 1 below). Figure 1 Shows the ordering of water molecules surrounding a hydrophobic molecule. Green lines indicate hydrogen bonds.

13. The contribution of the hydrophobic effect to globular protein stability has been estimated empirically both by measuring the thermodynamics of transfer of model compounds (e.g. blocked amino acids, cyclic peptides...) from organic solvents to water, and by site directed mutagenesis studies on proteins. The number arrived at is usually given as a function of the change in the solvent accessible non-polar surface area upon going from the unfolded to the folded state. The decrease in entropy (i.e. negative  S) of the solvent means that dissolving the non-polar molecule in water is thermodynamically unfavourable (i.e. positive  G). Hence the driving force of protein folding is thought to be the hydrophobic effect i.e. the hydrophobic side chains aggregate excluding water molecules as the protein folds. The resulting increase in entropy of these water molecules gives rise to a large positive  S causing the  G of folding to be negative i.e. thermodynamically favorable. Note that the entropy of the polypeptide itself decreases on folding which will counteract the increase in  S due to the waters.

14. Model compound studies predict that the hydrophobic effect of exposing one buried methylene group to bulk water is 0.8 kcal/mol. The site directed mutagenesis studies yielded a larger number with greater statistical variation: the average hydrophobic effect estimated by SDM for a buried methylene group is about 1.3 kcal/mol. However, when the SDM results for methylene were plotted against the size of the cavity created by the residue substitution, and extrapolated to zero, the result at zero cavity size is 0.8 kcal/mol - in agreement with the value found for the transfer of model compounds from octanol to water. In the SDM studies, cavities created by residue substitution have an additional destabilizing effect: the loss of favourable VDWs interactions (as compared to the wild-type). Thus, the "hydrophobic effect" measured by SDM includes both an entropic component due to solvent ordering and a (primarily) enthalpic component due to loss of VDWs contacts within the protein.

15. Such an SDM study of T4 lysozyme replaced the 80% buried Ile3 residue by Val the loss of this methyl group gave rise to a decrease in stability of 0.6 kcal/mol (corrected to 100% burial). This is smaller than expected (c.f. 0.8 kcal/mol for methylene) and suggests that the mutation introduced some smaller stabilizing influence, perhaps such as the alleviation of strain within the protein. In barnase, 15 mutants were constructed in which a hydrophobic interaction was deleted (V10A, V36A, V45A, I4A, I25A, I51A, I55A, I76A, I109A, I4V, I25V, I51V, I55V, I76V & I109V). The finding was a strong correlation between the degree of destabilization (which ranges from 0.60 to 4.71 kcal/mol) and the number of methyl or methylene side chain groups surrounding the methyl or methylene group that was deleted (r = 0.91). Correlation between the number of side chain methylene and methyl groups, in a radius of 6 Å of the group deleted from wild-type, and the changes in the free energy of unfolding for mutations of hydrophobic residues in barnase.

16. The average free energy decrease for removal of a completely buried methylene group was found to be 1.3 Kcal/mol. The number varies with the experiment. The number is also additive, such that Ile or Leu to Ala can destabilize a protein by up to 5 kcal/mol. (Remember that many proteins are stable by <10 kcal/mol, so two deletions such as this would be enough to destabilize a protein completely).