Transcript The Hydrophobic Effect. Hydrophobic Interactions: These are very

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 34-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.
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 ?”.
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.
Hydrophobicity Scale Values
Amino Acid
Engleman- Hopp- KyteEisenbergJanin Chothia
Steitz
Woods Doolittle
Weiss
PHE
-3.7
-2.5
2.8
0.5
0.0
0.61
MET
-3.4
-1.3
1.9
0.4
-0.24
0.26
ILE
-3.1
-1.8
4.5
0.7
0.24
0.73
LEU
-2.8
-1.8
3.8
0.5
-0.12
0.53
VAL
-2.6
-1.5
4.2
0.6
0.09
0.54
CYS
-2.0
-1.0
2.5
0.9
0.0
0.04
TRP
-1.9
-3.4
-0.9
0.3
-0.59
0.37
ALA
-1.6
-0.5
1.8
0.3
-0.29
0.25
THR
-1.2
-0.4
-0.7
-0.2
-0.71
-0.18
GLY
-1.0
0.0
-0.4
0.3
-0.34
0.16
SER
-0.6
0.3
-0.8
-0.1
-0.75
-0.26
PRO
0.2
0.0
-1.6
-0.3
-0.9
-0.07
TYR
0.7
-2.3
-1.3
-0.4
-1.02
0.02
HIS
3.0
-0.5
-3.2
-0.1
-9.94
-0.40
GLN
4.1
0.2
-3.5
-0.7
-1.53
-0.69
ASN
4.8
0.2
-3.5
-0.5
-1.18
-0.64
GLU
8.2
3.0
-3.5
-0.7
-0.90
-0.62
LYS
8.8
3.0
-3.9
-1.8
-2.05
-1.1
ASP
9.2
3.0
-3.5
-0.6
-1.02
-0.72
ARG
12.3
3.0
-4.5
-1.4
-2.71
-1.8
0.70
0.10
-0.47
0.10
-2.4 -0.45
-0.98
-0.51
THRESHOLD VALUES
Hydrophobic
-1.4
-0.75
Hydrophilc
1.85
1.65
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.
37
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
 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).
(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.
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 nonpolar group. As the temperature decreases the strength of the
hydrophobic interaction decreases: this is the opposite effect to that of Hbonds, 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.
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.
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 Hbond 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.
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.
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.
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.
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).