The Hydrophobic Effect.

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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.
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Consider a simple hydrocarbon (propane) C3H8
introduced into water (i.e. transferred from
pure liquid). C3H8(l) ? C3H8(aq)
?Ho298 -8kJ/mole (favorable !!)
?So298 -80J/oK-mole (unfavorable) ?Go298
16kJ/mole (unfavorable)   The formation
of oil drops is an entropy-driven process. The
question is Why ?.
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There are numerous scales of amino acid
hydrophobicity. Typically the hydrophobicity is
measured in terms of the free energy of transfer
(?Gtr) of the group of interest from aqueous
solution to a non-polar solvent, often octanol.
In general, a good correlation is found between
?Gtr values and other measures of hydrophobicity,
as well as the accessible surface area of the
amino acid side chains.
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Correlation hydrophobicity with accessible
surface area.
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.
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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 H2O 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 ? Slt0. 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).
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(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.
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The enthalpy contribution of the hydrophobic
interaction is approx. 0 around 20C, i. e. room
temperature, whereas the entropy contribution
becomes 0 around 140C. 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 (?Gtr) of the group of interest from
aqueous solution to a non-polar solvent, often
octanol. In general, a good correlation is found
between ?Gtr values and other measures of
hydrophobicity, as well as the accessible surface
area of the amino acid side chains.
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• Non-covalent Forces in Proteins
• 1.Hydrogen bonds
• Salt-bridges
• Dipole-dipole interactions
• Van der Waals forces
• 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.

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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.
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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.
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.
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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.
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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.
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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 lt10 kcal/mol, so two deletions such
as this would be enough to destabilize a protein
completely).