Open Journal of Bi op hysi cs, 2011, 1, 1-7
doi:10.4236/ojbiphy.2011.11001 Published Online October 2011 (http://www.SciRP.org/journal/ojbiphy)
The Rise and Fall of the Hydrophobic Effect in Protein
Folding and Protein-Protein Association and Molecular
Recognition
Arieh Ben-Naim
Department of Physi cal C hemistry , The Hebrew University of Jerusalem,
Edmond J. Safra Campus, Givat Ram, Jerusalem, Israel
Received September 14, 2011; October 10, 2011; October 17, 2011
Abstract
In the beginning everything was explained in Biochemistry in terms of hydrogen-bonds (HB). Then, the de-
vastating blow, known as the HB-inventory argument came; hydrogen bonding with water molecules com-
pete with intramolecular hydrogen-bonds. As a result, the HBs paradigm fell from grace. The void that was
created immediately filled by Kauzmann’s idea of hydrophobic (
H
O

) effect which reigned supreme in bio-
chemical literature for over 50 years (1960-2010). Cracks in the HB-inventory argument on one hand, and
doubts about the adequacy of Kauzmann’s model for the
H
O

effect, have led to a comeback of the HBs,
along with a host of new hydrophilic (
H
I

) effects. The
H
O

effects lost much of its power—which it
never really had-in explaining protein folding and protein-protein association. Instead, the more powerful
and richer repertoire of
H
I

effects took over the reins. The
H
I

interactions also offered simple and
straightforward answers to the problems of protein folding, and protein-protein association.
Keywords:
Protein Folding, Protein Association, Molecular Recognition, Hydrophobic, Hydrophilic
Interactions
1. Introduction
This article tells the story of the rise and fall of the hy-
drophobic (
H
O

) effect in protein folding and pro-
tein-protein association. Parallel to this story is the story
of the
fall and rise of the (hydrophilic)
H
I

effects.
The two stories are intertwined; the fall of one led to the
rise of the second. The fall of the second led to the
comeback of the first. These two stories have also an
important
moral regarding the mechanism of evolution,
survival and extinction of ideas in the biochemical lit-
erature, and perhaps in science, in general. The
moral is
not discussed in this article.
A convenient point to begin with the story is Pauling’s
book “The Nature of the Chemical Bond” [1,2]. In the
first two editions of the book Pauling discussed the HB,
but no mention of proteins or nucleic acids. In the second
edition, the chapter on HBs (Chapter IX) ends with some
estimates of the HB energies and HB distances. The third
editions contain two new sections on HBs in proteins and
HBs in nucleic acids.
Following the works of Anson and Mirsky [3] and
Mirsky and Pauling [4] on the denaturation of proteins,
and latter works of Pauling and Corey [5-8], the role of
HBs in stabilizing the native form of proteins became the
dogma [9]. The HBs, with bond energies of the order of
24 kJ/mol, which provided explanation for many
anomalous properties of water, also took over the main
“cohesive forces needed for the organization of native
proteins” [9].
The first cracks in the HB-dogma came from the re-
alization that though the HB energ y is the order of 24
kJ/mol, its formation in aqueous solution must have a far
smaller effect on the “driving force” for the process of
protein folding. The argument apparently started with the
work of Schellman [10,11] summarized by Kauzmann
[12] and eventually was encapsulated in Fersht’s HB-
inventory argument [13]. The argument seems simple,
straightforward and convincing. Write the stoichiometri-
cal reaction between a donor and an acceptor of a HB in

2
A. BEN-NAIM
the form
ECOww HNS
ECOHNSw w
 
 


(1)
where
E and S stand for an enzyme and a substract, re-
spectively, but can be any two molecules or parts of the
same molecule, and
w is a water molecule. Equation (1)
suggests that in the process of formation of a
direct hy-
drogen bond between a donor (here amine group) and an
acceptor (here a carbonyl group),
two HBs are broken on
the lhs of the equation and
two HBs are formed on the
rhs of the equation. Therefore, ignoring the differences in
the various HB energies between the various pairs (car-
bonyl-water, amine-water, carbonyl-amine and wa-
ter-water), we can conclude by simply
counting , that the
net effect of the formation of a direct HB is negligibly
small. As Kauzmann summarized this argument:
“
Hydrogen bonds , taken by themselves , give marginal
stability to ordered structures , which may be enhanced
or disrupte d by interactio ns o f side chains. ”
We use here the term “driving force” in the sense of
Δ
G < 0 as commonly used in the literature.
If HBs do not provide the main “driving force” for
protein folding, what factors do provide those “driving
force?” [14,15]. This apparently conceptual vacuum was
filled by the
H
O

effect in (1959). The
H
O

effect
was known long before Kauzmann applied it to the
problem of protein folding [9].
It was applied success-
fully to explain surface tension of certain aqueous solu-
tions of organic molecules, micelle formation and mem-
branes. All these phenomena involve molecules having
two moieties; a hydrophobic part, one “feared” water and
tries to avoid it, the second “loved” water and mingled
with water comfortably. As Tanford and Reynolds
quoted from a personal communication with Kauzmann,
the idea of the
H
O

effect was hovering “in the air” for
long a long time [9].
In a classical review article “Some Factors in the In-
terpretation of Protein Denaturation,” Kauzmann applied
the idea of the
H
O

effect to protein folding [12]. For
this purpose he coined the term
H
O

-bond, and specu-
lated that this “bond” could be the more important factor
in the stabilization of the native structure of protein.
Kauzmann’s idea was very simple. It was known that
the Gibbs energy of transferring a small non-polar solute
such as methane or ethane, from water into an organic
liquid involves a large negative change in Gibbs energy.
This is the same “driving force” that drives the formation
of micells and membranes in aqueous solutions.
Kauzmann also noticed that there are about one third of
amino-acid side-chains which are
H
O

, and most of
these find themselves in the interior of the folded protein.
If one can take the process of transfer, Figure 1(a), to
represent the process of transfer of side chain, Figure
1(b), then we can estimate that a protein of about 150
amino acid has about 50
H
O

groups, and if each of
these contributes between –12 and –16 kJ/mol, we get a
very large “driving force” for the folding process.
Kauzmann’s idea was brilliant. It captured the imagi-
nation of many scientists including mine. Add to it the
fact that the process of transferring of a non-polar solute
from water into an organic liquid is “
entropy driven ” .
Add to it that
entropy is a mysterious concept [16], not
well understood, and you get a “driving force” which is
enshrouded with an aura of mystery. Given all these facts,
many authors could have claimed that the
H
O

effect is
the
most important “driving force” in protein folding,
without taking any risk of being proved wrong!
How can anyone prove anything on such a complex
process as protein folding [15]. Carried out in poorly
understood solvent [14], involving a mysterious “entropy
driven” concept [10]?
It is not surprising therefore, that the dominance of the
H
O

effect has prevailed for over half a century. The
fact that
H
O

groups are in the interior of the protein ,
and the fact that the transfer of
H
O

molecules from
water to an organic liquid is large and negative are unde-
niable. The former lends credibility to Kauzmann’s
model, while the latter provides the large negative Gibbs
energy change. Although the molecular source of these
large negative Gibbs energy changes were not clear, one
can always say: “That is an entropy effect” and that is
more than enough to silence any objections. I would not
go that far as the von Neumann’s famous statement that
“No one knows what entropy really is” [17,18], however
knowing or not knowing what entropy is, the mere in-
voking of
entropy in the “driving force”, confers re-
spectability and authority to the person who use it.
(a) (b)
Figure 1. (a) The process of transferring a methane mole-
cule from water into an organic liquid. (b) The process of
transferring a methyl group from water into the interior of
the protein.

A. BEN-NAIM
3
2. The Cracks in the
H
O

Dogma
As I have said in the previous section, the
H
O

dogma
is still alive and thriving. One can find statements about
the “dominance” of the
H
O

effect in protein folding
even in the most recent reviews and textbooks. Never-
theless, a few whispers of doubts have been heard during
the past 20 years.
In 1980, in the preface of my book “Hydrophobic In-
teractions,” I wrote [19]:
“In spite of my researches in this field over almost 10
years, I cannot confirm that there is at present either
theoretical or experimental evidence that unequivocally
demonstrates the relative importance of the
H
O

inter-
actions over other types of interactions in aqueous solu-
tions.”
My doubts were based on lack of evidence in favor of
the contention that the
H
O

effect is the most impor-
tant effect in the “driving force” for protein folding. How
can one claim that one factor is more important, or most
important when one does not have a full
inventory of all
the factors involved in protein folding? Remember that
Kauzmann’s paper was on “some factors in the interpre-
tation of protein denaturation”—not on
all factors in-
volved. No one knew what were all the factors especially
those that are solvent-induced. The only factor that could
have competed with the
H
O

effect was the HB, but
the HB-inventory argument, debilitated the effect of the
HBs in aqueous media, and rendered them powerless in
explaining the driving force for protein folding.
Kauzmann’s model of inference from transferring of
molecules from water to organic liquid, and the fact that
most
H
O

groups are found in the interior of the pro-
tein were so convincing that mere expression of doubts
could not have rattled the dominance of the
H
O

dogma. One needs more than doubts. One needs facts!
“Lack of evidence” for an idea cannot be used as evi-
dence against that idea.
This was the main motivation for the examination of
the entire question of the solvent-induced effects on the
protein folding and protein-protein association that I un-
dertook late in the 1980s. The results of this examination
were stunning; initially to me, then slowly diffusing into
the literature.
First, it was found that the HB-inventory argument
was fundamentally faulty [20]. Second, Kauzmann’s
model, appealing as it was, for over 50 years was found
irrelevant to the protein folding process [21-25]. Finally,
a logical pitfall: The fact that
H
O

groups are found in
the interior of the protein cannot be used as an argument
in favor of the role of the
H
O

effect in protein folding.
I shall briefly explain here in qualitative terms each of
the abovementioned findings. More can be found in ref-
erences [14] and [15]. Recall that the HB-inventory ar-
gument was based on the stoichiometric Equation (1).
The mere counting of the number of HBs on each side of
the equation led to the dismissal of the role of direct hy-
drogen bonding to the driving force. The first serious
challenge to the HB-inventory argument was expressed
in 1990 [20]. It was shown that the very writing of the
stoichiometric equation in the form (1) is faulty for two
reasons:
1) What one loses on the left hand side are not HB
en-
ergies but solvation Gibbs energies of the
H
I

groups.
2) Whatever the water molecules do when they are re-
leased from the solvation sphere of the two
H
I

groups
is irrelevant to the driving force. These water molecules
“flow” from the solvation sphere into the pool of water at
constant chemical potential. Therefore, they cannot con-
tribute anything to the driving force. The stoichiometric
reaction must be written instead in the form



s
olvated solvated
solvated
ECOHNS
ECO HNS
 
  (2)
Here, each of the solutes (or the groups) involved in
the formation of a HB is solvated by the water molecule.
In this form, the HB-inventory argument does not exist.
Therefore, the foundations on which the
H
O

has risen
have now been demolished. This particular
H
I

effect
was estimated to contribute somewhat between –4 and
–6 kJ/mol to the driving force of protein folding, for each
intramolecular HB formed between two “arms” of the
H
I

groups [20].
Second, the analysis of all the solvent-induced factors
revealed that Kauzmann’s model does not feature in the
“driving force” for the process of protein folding [22,23].
Instead of the Gibbs energy of solvation of a
H
O

molecule in water, the conditional solvation Gibbs en-
ergy of a
H
O

group features in the “driving force.”
These Gibbs energies are very different from the Gibbs
energies of solvation in water. The main reason is that a
H
O

group attached to the backbone (BB) of the protein
is surrounded by water molecules which are perturbed by
the BB.
Thus, not only the basis on which the
H
O

model
was built upon was demolished, but the Kauzmann’s
H
O

-model itself was now shown to be inadequate
[14,15].
Finally, the fact that the
H
O

groups are in the inte-
rior of the protein does not necessarily mean that the
H
O

effect is the “driving force” for protein folding.
“Can anything be more convincing?” Tanford and Rey-
nolds asked rhetorically [19]. Such an inference turned
out to be only an illusion [15]. This is exactly the same
argument invoked when two different ideal gases mixed
spontaneously. The mixing is a fact, but the mixing, in

4
A. BEN-NAIM
itself does not provide the “driving-force” for the mixing
process [18].
3. The Emergence of New
H
I

Effects
The consequences of the analysis of the solvent-induced
effects on protein folding, not only had undermined the
foundation on which the
H
O

dogma was erected, and
not only demolished the
H
O

dogma itself, but opened
the door to a host of new solvent-induced effects that
were never considered before. These effects involved
H
I

rather than
H
O

groups. The most important one,
and so far the most studied, was the pairwise
H
I

in-
teractions between pairs of
H
I

groups at a distance
between 4 - 5 Å. For this particular
H
I

interaction
there is overwhelming evidence that it is far stronger
than any of the
H
O

effects. The evidence comes from
theoretical estimates [14,15], simulations [26,27] and
experimental data [14,15,28].
There are also some esti-
mates of
H
I

effects involving three and four
H
I

groups. These are more powerful, but probably less fre-
quent [15].
The qualitative explanation of the pairwise
H
I

in-
teraction is quite simple. A
H
I

group is characterized
by a few “arms” along which HB may be formed. An
amine group on the BB of the protein has one arm, a
carbonyl group has two arms, a hydroxyl group three
arms, and a water molecule itself has four arms. When a
H
I

group is in water its arms are solvated by water
molecule. The Gibbs energy of solvation per one arm
was estimated to be of the order of –9.4 kJ/mol [14,15].
Note that this is quite different from a HB energy, as one
might have erroneously counted in the HB-inventory
argument.
When two such
H
I

groups approach each other to a
distance of about 2.8 Å they form a genuine HB. Thus,
the Gibbs energy balance is: loss of the solvation Gibbs
energy of two arms costs about 20 kJ/mol, and the for-
mation of a HB provides a HB energy of about –24
kJ/mol. Therefore, the net change in Gibbs energy for
this particular
H
I

effect at a distance of about 2.8 Å is
about –6 kJ/mol [14,15].
A more dramatic
H
I

effect was found at a distance
of about 4.5 Å, the same distance of the second nearest
neighbors in ice [25]. When two solvated arms approach
each other to this distance, and with the correct orienta-
tion, they do not lose their solvation Gibbs energy as in
the former
H
I

case. They also do not gain an HB en-
ergy. Instead, the mutual salvation Gibbs energy of the
pair of
H
I

groups increase by an amount which was
estimated to be between –10 and –12 kJ/mol.
The reason for such a strong
H
I

interaction is that
at this particular configuration the two arms of the two
H
I

groups can be bridged by a water molecule. It
should be stressed however that this effect is not due to a
formation of long-lived HB-bridge, as some have mis-
understood. Such a “permanent” bridge could provide
two HB energies, i.e. about –48 kJ/mol. The real effect is
a mutual solvation of the two arms of water molecules.
This effect involves HB energy, but also involves prob-
ability of finding a water molecule that can form a
HB-bridge between the two
H
I

groups. The most
direct evidence for the existence of such a
H
I

effect is
the second peak in the radial distribution function of pure
liquid water [14,15]. Other experimental evidence comes
from the relative solubilities of two isomers of the same
molecule, having two
H
I

groups at two different dis-
tances [15,24].
Because of the short range of the HB, there exists a
steep gradient of the potential of mean force between two
H
I

groups at a distance about 4.5 Å. This leads to a
strong forc e between the two
H
I

groups, a force
which plays a crucial role in the process of protein fold-
ing [15].
One can also think of other
H
I

interactions, one
involving one water molecule bridging three
H
I

groups, or two water molecules forming one bridge con-
necting two
H
I

groups. The former is strong, but rare,
the second might be more frequent but very weak.
Therefore, it is believed that the pairwise
H
I

interac-
tion at a distance of about 4.5 Å is the more important
among the
H
I

effects, hence probably the most im-
portant in the process of protein folding as well as in the
process of protein-protein association or protein binding
to DNA [25].
4. Dominant Forces and Driving Forces in
Protein Folding and in Protein-Protein
Association
In this section, I shall briefly describe how the
H
I

effects may be implemented to understanding the process
of protein folding, as well as protein-protein association,
protein binding to DNA, self assembly, drug design and
many other processes.
To begin with, we must first define the problem of
protein folding. In fact, there are several problems asso-
ciated with protein folding. The most general question
asked in this connection is that of the existence of a
“code” that translates from the sequence of amino acids
into a three dimensional structure. Following the works
of Anfinsen [29,30], and others on the spontaneous fold-
ing of a denatured protein into the original native struc-
ture, it was speculated that the “information” about the
folding pathway is already contained in the sequence of
amino acids. However, whether or not there exists such a

A. BEN-NAIM
5
“code” that translates from a sequence of amino acids to
a 3-D structure of the native protein, what makes the
sequence fold into the native structure is the set of
forces—not the thermodynamic “driving forces”—that
act of each group of the protein. As was shown in refer-
ence [15] among all possible forces acting on the groups
of the protein, the strongest forces are those between
H
I

groups mediated by the solvent.
The solvent-induced forces are probably the most im-
portant factor that governs the process of protein folding.
A discussion of the way these forces contribute to the
dynamics of protein folding was discussed in references
[15] and [25].
Here, I will describe only a few applications of the
solvent-induced intera c t io n s between
H
I

groups which
affect the solvation Gibbs energy of protein. These in
turn affects the solubility of proteins, the stability of the
native structure of the protein, the stability of dimers or
larger aggregates of proteins and molecular recognition.
4.1. Solvation and Solubility of Proteins
The high solubility of proteins in water is well known to
any biochemist. Yet the molecular reason for the solubil-
ity of protein is not less mysterious than the molecular
reasons for protein folding and self assembly of biologi-
cal macromolecules.
The effect of the
H
I

groups on the solubility of
protein was long recognized. However, what is less
known is that
H
I

interactions are decisive in deter-
mining the high solubility of the protein [15,31].
The high solubility of protein is not only the result of
the existence of
H
I

groups on the surface of the pro-
tein. Furthermore, it is very likely that pairs and higher
order correlations between
H
I

groups on the surface
of the protein contribute significantly, if not decisively in
making the proteins highly soluble.
4.2. Protein Folding
In an article entitled “The Problem of How and Why
Proteins Adopt Folded Conformations,” Creighton [32]
discusses the questions of How and Why as if they were
one. Clearly, if we knew all the forces acting on all the
atoms at each intermediate state of a specific protein, we
could answer the question of “Why” and thereby the
answer to the question of “How.” This answer is perti-
nent to that specific protein.
An analysis of all the contributions to the solvent-in-
duced effects on the driving forces for the process of
protein folding reveals that
H
I

interactions at a dis-
tance of about 4.5 Å are probably the strongest.
The first indication that such correlation might be im-
portant came from some experimental data published by
Haberfield, et al. [28]. These data were used to extract
the quantity we shall call the correlation between two
H
I

groups [25].
Soon, more data became available, as well as some
simulation of these
H
I

effects, [26,27], and a theo-
retical estimate of the strength of these effects [25]. All
these data, led to the conclusion that correlation between
two
H
I

groups (at the correct distance and orientation)
are quite significant, and their role in the process of pro-
tein folding should be taken more seriously.
The conclusion reached here has some overlaps with
the conclusion reached by Rose et al. [33]. Rose et al.
proposed an inversion of the “side-chain/backbone para-
digm.” We advocate the inversion of the
H
O

/
H
I

paradigm. Clearly, since most of the
H
I

are provided
by the backbone, it follows that the role of the backbone
should be more important than the role of the side chains.
In this sense there is an overlap between our proposal
and Rose, et al. proposal. However, the two proposals
are quite different. This is further discussed in reference
[15].
We now turn briefly to discuss the factors involved in
the process of protein folding, which are the real physi-
cal forces. These forces are the ones that guide the pro-
tein in folding along a narrow range of pathways, leading
to the native form in a relatively short time [15,25].
We start with Anfinsen’s classical work on the rena-
turation of ribonuclease [29,30]. Anfinsen found that a
denatured protein will fold spontaneously when the
proper environment for folding are restored (e.g. lower-
ing the temperature or removing a denaturating agent).
The folding occurs spontaneously without the need for
any additional information beyond that which is con-
tained in the sequence of amino acids.
Thus, the information required for the folding is
somehow inscribed in the sequence of the amino acids.
Without elaborating on the nature of the information
contained in the sequence of amino acids, it is clear that
this information is of the type of instructions. These in-
structions must be read first, then to be executed step by
step. The agent that does that job is the “proper environ-
ment”, and the most important component in this envi-
ronment is water.
It is believed that water not only “reads” the informa-
tion contained in the sequence of amino acids but also
translates the instruction into executable orders. These
“orders” are the forces that are exerted on each of the
atoms of the protein that causes the motion of the entire
protein towards the end product. Because of the statisti-
cal character of these forces the motion of the protein is
not along a unique deterministic route, but along a nar-
row range of routes or pathways.

6
A. BEN-NAIM
Most reviews on protein folding focus on the thermo-
dynamic “driving forces” rather than the forces them-
selves [34]. Recently, an analysis of the types of forces
acting on the protein, and more specifically on the sol-
vent-induced forces in protein folding, was undertaken
[15,25].
4.3. Self Assembly and Molecular Recognition
Binding, association and self assembly processes abound
in biological systems. These processes range from bind-
ing small ligands such as drugs to protein or to DNA,
association between proteins, as in hemoglobin and
self-assembly of a large number of subunits to form
macromolecules such as Tobacco mosaic virus.
There are essentially two puzzles associated with these
processes. The first is similar to the question of the sta-
bility of the folded protein. In the folding process, the
large number of conformational states of the unfolded
form tends to favor the denatured protein. To understand
the “driving force” for folding we need to find out the
factors that stabilize the native structure of the protein.
Similarly, in any association process there are far more
configurations to the separated units than to the bound
aggregates. Again, to understand the “driving force” for
the binding process, we must find out which factors are
responsible for the stabilization of the dimer or the oli-
gomer, relative to the separate monomers.
The second puzzle is similar to the preferential path-
ways of protein folding. It is concerned with the specific-
ity of the binding mode. There are many ways two
globular proteins can bind, yet only one specific binding
mode is stable. Specificity of binding is essentially the
same as molecular recognition. These phenomena are
relevant for diverse biochemical systems ranging from
binding drugs to protein (hence also to drug design),
binding of protein to DNA (controlling genetic expres-
sion), and the way the immune system works [35].
We have found that the
H
I

interactions could be
decisive in determining both the stability and the speci-
ficity of the mode of association between two proteins.
The finding that
H
I

interactions can change the
preferential binding site has far reaching consequences to
the problem of drug design, either for designing new
drugs or for modifying existing drugs to improve their
efficacy. Some specific examples were discussed re-
cently [36]. We shall not present these highly technical
examples here. The interested reader should consult the
article by Wang and Ben-Naim [36].
5. Conclusions
The paradigm change from the
H
O

to
H
I

effects
has brought us as close as one can hope for, to the solu-
tion of the problem of protein folding and self assembly
of proteins.
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