Chapter 10: Multiple equilibria, cooperativity and allostery.

10.1 Introduction
10.2 Analysis of multiple equilibrium problems: Adairs equation- A modelindependent approach.
10.3 Analysis of multiple equilibrium problems: The model-dependent approach
for a protein with two binding sites.
10.4 A simple protocol to write model-dependent binding isotherms by inspection.
10.4.1 Example 1: A protein with one binding site of ligand A and a second
site for ligand B with no cooperative interactions.
10.4.2 Example 2: A protein with two equivalent sites for ligand A and one
for ligand B, without cooperativity.
10.4.3 Example 3: A tetramer with 4 equivalent, independent binding sites
for ligand A.
10.5 Homotropic cooperativity
10.6 Heterotropic cooperativity
10.7 Coupling ligand binding to protein interactions
10.8 Infinite positive cooperativity, the Hill equation, and the width of a binding
isotherm.
10.9 Graphically displaying binding data
10.10 Example: The cooperative dimer
10.11 The distribution of protein species in solution
10.12 The magnitudes and origins of coupling free energies: models of allostery
10.12.1 Models of allostery

MWC model:
KNF model:
Current views:
10.13 Example: hemoglobin
Homotropic cooperativity and the MWC model:
Heterotropic cooperativity and the MWC model
Structural explanations of the observed thermodynamics:
Does the MWC model explain everything about hemoglobin cooperativity?
10.14 Example: The catabolite activator protein from E. coli
10.16 Summary

Chapter 10: Multiple equilibria, cooperativity and allostery.

10.1 Introduction
In this chapter we continue our treatment of the thermodynamics of ligand
binding, extending to systems in which there are coupled interactions at different sites on
the same protein. By definition, all these are examples of allostery, which describes all
systems in which binding at one location influences the function of a protein at another
distal site. Initially, this term was used primarily in reference to the binding of small
ligands to proteins, such as O2 binding to hemoglobin, or to enzymes in which, for
example, the interactions with substrates (binding or catalytic function) could be
modulated by the binding of a ligand at a site distant from the actual active site. These
systems all have in common the fact that the binding of a ligand at one site on a protein is
communicated through the protein to another location on the protein. The same is also
true in virtually all regulatory networks involved in controlling gene expression. The
presence of absence of a small molecule results in changing the one or a sequence of
protein-DNA interactions or protein-protein interactions, or functions such a
phosphorylation or acetylation. Virtually all biological signal transduction pathways
involve one or many instances in which an interaction at one site on a protein results in
changes at another site, leading to biological consequences. The quantitative description
of allostery and an understanding of the mechanisms by which a binding event is
communicated through a protein are central issues in biology and biochemistry.
The first point to appreciate is that allostery is a thermodynamic phenomenon,
measured using the same methodology we discussed in the previous chapters, and
quantified in terms of changes in standard state free energies, enthalpies and entropies.

The natural question of how to translate this in terms of protein structure and dynamics is
a separate question, albeit a very important one, though very far from being answered.
We will emphasize the thermodynamic treatment of systems involving multiple
equilibria.

10.2 Analysis of multiple equilibrium problems: Adairs equation- A modelindependent approach.

In the last chapter, we discussed situations in which the binding sites all have the
same affinity for the same ligand, e.g., one type of binding site, and there is no interaction
between the sites. Although this covers much of what one encounters in biochemical
systems, there are many interesting systems in which multiple ligands bind and the
occupancy of a binding site perturbs the subsequent binding of ligands to other binding
sites. This applies just as much to small molecule interactions with macromolecules as
with interactions between macromolecules (e.g., protein-protein or protein-DNA
interactions, for example). The thermodynamic analysis provides insights into all of
these systems, especially when coupled to structural or dynamics information.
The model-independent approach provides a mathematical description that has no
built-in assumptions about the number or type of binding sites. This is used, naturally,
when one does not have enough information about the system to guess one or a few
reasonable models. A model specifies the number of binding sites, whether they have the
same or different affinities for the ligand and whether the occupancy of one or more sites
influences the affinity for the remaining sites. A specific model will contain a
dissociation constant assigned to each particular binding. These are called

microscopic binding constants, and this is what one finds in most biochemical studies.
However, the model-independent approach is currently used (1) and it is helpful to point
out the distinctions of binding constants defined by model-independent vs modeldependent methods.
Adairs equation: Lets assume that we have a protein, P, with N binding sites
for a particular ligand, A. Nothing else is specified. The sites may be identical or each
different, and they may or may not interact with each other. We can define the following
phenomenological equilibrium expressions and the dissociation constants. Note that the
expressions are written in the direction of association, so G o = + RT ln K d since we are
using dissociation constants.

[ P][ A]
[ PA]
[ PA][ A]
K II =
[ PA2 ]

P + A R PA

KI =

PA + A R PA2
PA2 + A R PA3

K III =

[ PA2 ][ A]
[ PA3 ]

PA3 + A R PA4

K IV =

[ PA3 ][ A]
[ PA4 ]

(1.1)

etc
The expressions above are not true equilibrium expressions because the species that are
indicated as PA, PA2 , etc. are not unique chemical entities but represent groups, as we
discussed in Chapter 3 when grouping different protonation states as a single species. In
the current case, PA , for example, designates the protein with one ligand bound, but that
ligand can be in any binding site. If we have 4 binding sites, then there are four
distinguishable species if the binding sites themselves are distinguishable.

This is

illustrated in Figure 10.1 which shows the various species collected together with 0, 1, 2,

3 and 4 ligands bound to a protein with 4 different binding sites for the same ligand.
There are 1, 4, 6, 4 and 1 species, respectively for 0, 1, 2, 3 and 4 ligands bound to the
protein with 4 binding sites (Figure 10.1). In the general case of n ligands bound to a
protein with N distinguishable sites, there are P ( n, N ) distinct species, where P ( n, N ) is
defined by the binomial distribution (see Appendix 2).
P ( n, N ) =

N!
( N n)!n !

(1.2)

The dissociation constants defined in equations (1.1) are called macroscopic or

statistical dissociation constants. They are distinguished from microscopic or intrinsic
dissociation constants which are defined in terms of chemically defined species, which

is defined in terms of a specific biochemical model.

Figure 10.1: A protein with four different binding sites for the same ligand. The
species present in solution are the free ligand plus each of the protein species
pictured on the right. These can be grouped together to define phenomenological or
macroscopic dissociation constants, K I , K II , K III , and K IV .

Lets say we measure the amount of bound ligand per protein molecule to define the
binding isotherm, vs [ A] , and would like to analyze the data. Lets define the binding
isotherm in terms of the groups of protein speices with 0, 1, 2, 3 etc ligands bound.

[ A]bound [ PA] + 2[ PA2 ] + 3[ PA3 ] + ... + N [ PAN ]

=
[ P ]total [ P ] + [ PA] + [ PA2 ] + [ PA3 ] + ... + [ PAN ]

(1.3)

The coefficients in the numerator are there to account for the fact that each molecule of
PA2 contains 2 equivalents of ligand A, PA3 contains 3 equivalents of ligand A, etc. The

numerator is the sum of the bound ligand concentrations, so these coefficients are
included. In the denominator, we are summing the concentrations of the protein species,
regardless of the number of ligands bound, and the coefficients are absent. We now
substitute from the definitions of the dissociation constants in equations (1.1).
[ A]
[ A] [ A]
[ A] [ A] [ A]
[ A]N
+2
+3
+ ... + N
K
K I K II
K I K II K III
K I K II ...K N
= I
[ A] [ A] [ A] [ A] [ A] [ A]
[ A]N
+
+
+ ... +
1+
K I K I K II K I K II K III
K I K II ...K N
or

(1.4)

[ A]
[ A]2
[ A]3
[ A]N
+2
+3
+ ... + N
K
K I K II
K I K II K III
K I K II ...K N
= I
2
3
[ A]N
[ A] [ A]
[ A]
+ ... +
+
+
1+
K I K II ...K N
K I K I K II K I K II K III
he experimental data can be analyzed by determining the best fit to equation (1.4) with
the minimum number of parameters (dissociation constants). This will define the
binding stoichiometry (number of binding sites) but otherwise further interpretation of
the biochemical meaning contained within the dissociation constants will require
interpretation in terms of a specific model.

10.3 Analysis of multiple equilibrium problems: The model-dependent approach

for a protein with two binding sites.

We will now obtain the binding isotherm for a protein with two binding sites for
the same ligand. We will define a specific model that the two binding sites are
distinguished by two different dissociation constants, K1 , K 2 , and there is no
cooperativity between the sites. The absence of cooperativity means that the binding
constant for site 1 is the same regardless of whether site 2 is occupied. This model is
summarized in Figure 10.2.

Figure 10.2: The model of an asymmetric dimer without cooperativity- a protein

with two different binding sites for a ligand and no interaction between the two
sites. Binding to the two different sites is designated as PA vs PA .

The measured binding isotherm will be the following.

[ PA] + [ AP ] + 2[ APA]
[ P ] + [ PA] + [ AP ] + [ APA]

(1.5)

Substituting from the definitions of the microscopic dissociation constants in Figure

10.2, we now get.

[ A] [ A]
[ A] [ A]
+
+2
K
K2
K1 K 2
= 1
[ A] [ A] [ A] [ A]
+
+
1+
K1 K 2 K1 K 2

(1.6)

If we knew the system we were studying had 2 different binding sites, say with high and
low affinities for the ligand, we would use equation (1.6) to fit to the experimental data
and obtain the best values for the two intrinsic dissociation constants, K1 and K 2 .
Now, lets change the model to one in which the two binding sites are identical,
but there is still no cooperativity between the sites. This is summarized in Figure 10.3
and simply define K1 = K 2 K d .

Figure 10.3: The model of a symmetric dimer with no cooperativity.

The binding isotherm can now be written as follows.

[ PA] + [ AP ] + 2[ APA]
[ P ] + [ PA] + [ AP ] + [ APA]

[ A] [ A]
[ A] [ A]
+
+2
K
Kd
Kd Kd
= d
[ A] [ A] [ A] [ A]
+
+
1+
Kd Kd Kd Kd

(1.7)

[ A]
[ A] [ A]
+2
Kd
Kd Kd
=
[ A] [ A] [ A]
+
1+ 2
Kd Kd Kd
2

The coefficient that comes from converting from protein to bound ligand concentration
are colored in red, and the coefficients due to collecting equivalent terms, each derived
from an identical species, are colored in blue.
We can compare the final expression in equation (1.7) with the modelindependent expression for a dimer from equation (1.4).

[ A]
[ A]2
+2
K
K I K II
= I
[ A] [ A]2
+
1+
K I K I K II

(1.8)

From this we can see that the statistical binding constants and the intrinsic binding
constants are related in the following manner.
KI =

Kd
2

and

K II = 2 K d

(1.9)

The two statistical binding constants, K I and K II are different even though the intrinsic
binding constants for the two sites are equivalent. This is because we have defined the
statistical binding constants in terms of groups of species. In going from two empty sites

10

to one occupied site, there are two different microscopic states that are equivalent
( AP and PA ), so there is an apparent gain in configuration entropy when we use this
definition of the dissociation constant. This results in the apparently stronger affinity for
the first binding event (smaller dissociation constant) than for the second binding event,
when the number of equivalent species decreases from 2 to 1. This is pictured in Figure
10.4.

Figure 10.4: The relationship between the statistical binding constants and the
intrinsic binding constant for a symmetric dimer.
10.4 A simple protocol to write model-dependent binding isotherms by inspection.

We will now concern ourselves exclusively with model-dependent binding

isotherms since this is what most biochemists utilize. We can use the procedures that
were employed to obtain the binding isotherm for the symmetric dimer, equation (1.7) to
any case, no matter how complicated. Instead of going through all the algebra, however,
11

there is a pattern that is already evident in equation (1.7) which will allow us to write
down the binding isotherm for more complicated cases just by inspection of the model.
The protocol is the following:
1. Define each of the protein species that is present in the solution:
P, PA, PA2 , PB etc
2. Write the expression for the binding isotherm in terms of the concentration of
these species, including the coefficient to account for multiple ligands on each protein
species: [ P],[ PA],[ PA2 ] or 2[ PA2 ],[ PB] etc.
3. Substitute for each term using the following pattern:

4. Add the coefficient in front of each term equal to the number of equivalent
species.
We can look at a few examples to illustrate this. In all these examples there is no
cooperativity between binding sites. This will be discussed in the next section.

12

10.4.1 Example 1: A protein with one binding site of ligand A and a second site for
ligand B with no cooperative interactions.

This is illustrated in Figure 10.5.

Figure 10.5: A protein with one binding site each for ligand A and B, with no
cooperativity.

The binding isotherm for ligand A is

A =

[ PA] + [ BPA]
[ P] + [ PA] + [ BP] + [ BPA]

(1.10)

and the binding isotherm for ligand B is

B =

[ BP] + [ BPA]
[ P] + [ PA] + [ BP] + [ BPA]

(1.11)

There are no species with more than one of either ligand, and there are no species with
multiple equivalent species, so the final isotherms are written as follows.

13

[ A] [ B ]
[ A] [ A] [ B]
+
1 +

K A KB
K A K A KB
=
A =
[ A] [ B] [ A] [ B] [ B] [ A] [ B]
1+
+
+
1+
1+
+
K A K B K A K B K B K A K B
[ A]
[ B]
KA
KB
and similarly B =
A =
[ A]
[ B]
1+
1+
KA
KB

(1.12)

Since there is no communication between the two sites, the binding of A and B are
independent of the presence or absence of the other ligand. The form of the binding
isotherm is that expected for a single binding site.
10.4.2 Example 2: A protein with two equivalent sites for ligand A and one for
ligand B, without cooperativity.

The binding isotherm for ligand A is

A =

[ PA] + 2[ PA2 ] + [ BPA] + 2[ BPA2 ]

[ P ] + [ PA] + [ PA2 ] + [ BP ] + [ BPA] + [ BPA2 ]

(1.13)

Substitute the appropriate terms, plus the coefficient 2 in front of each of the terms for
[PA] and [ BPA] since there are two equivalent species, due to the two equivalent binding

sites for ligand A.

[ A]
[ A] [ A]
[ A] [ B]
[ A] [ A] [ B]
+2
+2
+2
KA
KA KA
K A KB
K A K A KB
A =
[ A] [ A] [ A] [ B]
[ A] [ B] [ A] [ A] [ B]
1+ 2
+
+
+2
+
K A K A K A KB
K A KB K A K A KB
2

(1.14)

which simplifies to the expected form for two equivalent, independent binding sites.
[ A]
KA
A =
[ A]
1+
KA
2

(1.15)

14

Similary, the isotherm for ligand B is

B =

[ BP] + [ BPA] + [ BPA2 ]

[ P] + [ PA] + [ PA2 ] + [ BP] + [ BPA] + [ BPA2 ]

[ B]
[ A] [ B] [ A] [ A] [ B]
+2
+
KB
K A KB K A K A KB
B =
[ A] [ B] [ A] [ A] [ B]
[ A] [ A] [ A] [ B]
1+ 2
+
+
+2
+
K A K A K A KB
K A KB K A K A KB

(1.16)

[ B]
KB
B =
[ B]
1+
KB
10.4.3 Example 3: A tetramer with 4 equivalent, independent binding sites for
ligand A.

The binding isotherm is equal to

[ PA] + 2[ PA2 ] + 3[ PA3 ] + 4[ PA4 ]

[ PA] + [ PA2 ] + [ PA3 ] + [ PA4 ]

(1.17)

Substitute the appropriate terms plus the coefficients for the number of equivalent
species. The coefficients are given by the binomial distribution in Figure 9.26.
[ A]
[ A] [ A]
[ A] [ A] [ A]
[ A] [ A] [ A] [ A]
+ 2(6)
+ 3(4)
+4
KA
KA KA
KA KA KA
KA KA KA KA
=
[ A]
[ A] [ A]
[ A] [ A] [ A] [ A] [ A] [ A] [ A]
+6
+4
+
1+ 4
KA
KA KA
KA KA KA KA KA KA KA
4

(1.18)

This simplies, fortunately, also to the following.

15

[ A]
[ A] 3
[1 +
]
KA
KA
=
[ A] 4
[1 +
]
KA
4

(1.19)
[ A]
KA
=
[ A]
[1 +
]
KA
4

Once again, as in the previous cases, we end up with the equivalent of the HendersonHasselbach equation. This is because in each of these examples we have no interaction
between the sites, so it does not matter of other ligands are bound or not and it does not
matter whether the binding sites are grouped in 2 per protein or 4 per protein, etc.
The general expression for a protein with N independent and equivalent sites is
[ A]
KA
=
[ A]
[1 +
]
KA
N

or

(1.20)

[ A]
KA

= =
N [1 + [ A] ]
KA
10.4.3 Multiple classes of binding sites for the same ligand.

If one has a protein with sites which have different intrinsic affinities for the
same ligand, then one gets a form that is simply given by the sum of the binding to the
different classes of binding sites. Lets say a protein has N s strong binding sites ( K s ) and
N w weak binding sites ( K w ) for the same ligand. Again, we specify no cooperativity, so

16

we can simply sum the binding to the two classes of binding sites to obtain the final
experimental binding isotherm.

= s + w
(1.21)
[ A]
[ A]
Nw
Ks
Kw
+
= =
[ A]
[ A]
[1 +
] [1 +
]
Ks
Kw
Ns

If one had binding data for a system where it was thought this was the appropriate model,
one would fit the data to equation (1.21). Note that with just to classes of binding sites,
there are already 4 independent parameters that one can use to obtain the best fit:
N s , K s , N w and K w .
10.5 Homotropic cooperativity

When the binding of a ligand alters the affinity for subsequent binding of the
same ligand to unoccupied sites, this is called homotropic cooperativity. This is
distinguished from heterotropic cooperativity, which involves the interactions between
the binding of different ligands, discussed in the next section. The best example of
homotropic coopertivity is the binding of oxygen to hemoglobin, which will be discussed
in more detail in Section 10.13. Positive cooperativity is when the binding of the first
ligand enhances the binding of the next ligand. Negative cooperativity is when the
binding of the first ligand weakens the affinity of the next ligand to bind.
We can illustrate with the example of a cooperative dimer, a protein with two
identical binding sites for ligand A. Occupancy of either binding site alters the affinity of
the protein for the ligand binding at the second site. This is the situation in Figure 10.6.

17

Figure 10.6: A cooperative dimer. Two identical sites for the same ligand. Binding to
either site alters the affinity at the remaining site. The intrinsic dissociation
constants are defined, as is their ratio.

The ratio of the intrinsic dissociation constants is defined as (see Figure 10.6). When

= 1 , there is no cooperativity, whereas when < 1 , the affinity increases for the second
ligand to bind (smaller dissociation constant), defined as positive cooperativity. Negative
cooperativity is when > 1 . A free energy diagram for this situation is shown in Figure
10.7. Three different situations are indicated: no cooperatiivty (left), positive

cooperativity (middle) and negative cooperativity (right). The influence of the first
o
ligand is quantified by the coupling free energy, Gcoupling
. From the definition of , it
o
o
= RT ln . When < 1 , then Gcoupling
< 0 , meaning that the drop
follows that Gcoupling

18

in the standard state free energy is more negative for the binding of the second ligand,
i.e., the affinity is increased.

Figure 10.7: Free energy diagram for a dimer with two identical binding sites for
ligand A. On the left: no cooperativity; middle: positive cooperativity; right:
negative cooperativity. The levels indicate the sum of the standard state chemical
potentials of the species indicated. The directions of the reactions are towards
associations, but the binding constants are intrinsic dissociation constants.
10.6 Heterotropic cooperativity

We can treat heterotropic cooperativity in the same was as we did homotropic

cooperativity. Lets consider a protein with one binding site for ligand A and a second
binding site for ligand B. If there is cooperativity, then the binding of A will alter the
binding of B. It follows from the thermodynamics that if the binding of A influences the
binding of B, then the reverse must also be true, and by the same extent. This is a called
reciprocity. We can see why this is the case by examining the diagram in Figure 10.8.

19

Figure 10.8:Heterotropic cooperativity. The intrinsic dissociation constants are

defined, along with their ratios.

The series of binding reactions shown in Figure 9.33 defines a thermodynamic cycle.
Therefore, since free energy is a state function, we can take either way around to go from
the unliganded protein to the fully liganded protein ( P BPA )
G1o + G4o = G2o + G3o

Since G1o = RT ln K1 , etc., it follows that

K1 K 4 = K 2 K 3

(1.22)

(1.23)

or
K3 K 4
=

K1 K 2

(1.24)

20

Figure 10.9 shows the free energy diagram for two situations: the non-cooperative case,

when = 1 , and the case of positive cooperativity when < 1 , meaning that the present
of ligand A enhances the binding of ligand B. Reciprocity tells us that the reverse must
also be true. The coupling parameter, , is related to the coupling free energy,
o
Gcoupling
= RT ln , as was the case for homotropic cooperativity. Furthermore, the free

energy diagram makes it clear that the change in the standard state free energy of binding
of ligand A due to the presence of ligand B must be exactly the same as the change in the
o
binding free energy of ligand B due to the presence of A: Gcoupling
.

Negative heterotropic cooperativity is not pictured, but is essentially the same

o
> 0 , so the each ligand reduces the affinity of the second
except that > 1 and Gcoupling

ligand.

21

Figure 10.9: Free energy diagram showing heterotropic cooperativity. On the left is
the situation in which there is no coopertivity. The levels are the sums of the
standard state chemical potentials of the species shown, and the arrows are the
standard state free energy differences between the products, at the head of each
arrow, and the reactants, shown at the foot of each arrow.
10.7 Coupling ligand binding to protein interactions

A variation of heterotropic cooperativity is when the binding of a small ligand

changes the affinity between macromolecules, typically protein-protein or protein-DNA
interactions. Most signal transduction networks and transcription regulatory pathways
rely on this mechanism. We can look at one example of a ligand which can bind to a
protein and induce dimerization of the protein. We might diagram this in a biochemistry
textbook as in Panel A of Figure 10.10.

Figure 10.10: (A) A diagram indicting the conversion of an unliganded protein

which, upon binding a ligand, dimerizes. (B) Additional protein species that need to
be considered in developing a general model.

22

The various protein species present are shown in Figure 10.10, and the concentrations of
all species are interconnected through a set of equilibrium constants. We can use a free
energy diagram to show the various equilibria rather than write down each separately
(Figure 10.11). As we have done previously, the vertical arrows connect reactants and
products, and the levels are the sum of the standard state chemical potentials, since we
are dealing with equilibrium conditions.

Figure 10.11: Free energy diagram for the situation where a ligand binding
enhances dimer formation. On the left are defined the equiblria for the monomeric
species. In the middle, the situation where the protein can form a dimer but the
affinity of the protein for the ligand is the same for the dimer as it is for the
monomer. On the right is the situation where the dimer binds with greater affinity
to the ligand. By reciprocity, this means that the ligand binding enhances the
formation of the dimer. For simplicity, it is assumed that the binding to each site on
the dimer is the same.

23

In the middle part of Figure 10.11, the diagram shows the situation in which the ligand
affinity for the dimer is the same as for the monomer.
o
GAo = GDA

(1.25)
KA =

[ P][ A]
[ PA]

K DA =

[ P2 ][ A]
[ P2 A]

Because of this, the free energy of dimer formation must be the same.
KD =

[ P]2 [ PA]2 [ P][ PA]

=
=
[ P2 ] [ P2 A2 ]
[ P2 A]

GDo = RT ln K D

(1.26)

If we assume that the affinity of the dimeric protein is greater than for the monomeric
form of the protein, then this leads to the enhanced formation of dimmers.
o
if GDA
< G Ao

then G2o < GDo

(1.27)

From Figure 10.11, or by constructing a thermodynamic cycle, we can obtain the

quantitative relationships.
o
2( GDA
G Ao ) = (G2o GDo )

(1.28)

The term on the left of equation (1.28) is the difference in the binding free energies of the
ligand for the dimer and monomer protein species, and the term on the right is the
difference in the free energy of dimer formation in the presence and absence of the ligand
bound to the site.
10.8 Infinite positive cooperativity, the Hill equation, and the width of a binding
isotherm.

We now return to homotropic cooperativity, and consider the limiting situation in

which a number of equivalent sites (N) are coupled maximally so that the binding of the
first ligand results in a very large increase of the affinity of the remaining sites on the

24

protein for the ligand. This is called infinite positive cooperativity. We can get a good
sense of the consequences by using the example of a tetramer, with four equivalent
binding sites. After binding of the ligand to any of the four sites, the affinity goes up to
such an extent that the result is that the second, third and fourth site all become occupied,
regardless of the concentration of the free ligand. The consequence is that in solution, the
concentration of protein with 1, 2 or 3 ligands bound is negligible. Virtually all the
protein species will have either no ligands bound ( [ P ] ) or all the sites filled ( [ PA4 ] ).
This is pictured in Figure 10.12.

Figure 10.12: Infinite positive cooperativity between the four equivalent sites of a
protein. The presence of one ligand bound assures that the remaining sites will also
be occupied by ligands.

25

Lets say that the intrinsic dissociation constants each of the sites for the first, second,
third and fourth ligands are K1 , K 2 , K3 and K 4 . We can write the binding isotherm as
follows.

[ PA4 ]
[ P] + [ PA4 ]
(1.29)

[ A] [ A] [ A] [ A]
K1 K 2 K3 K 4
=
[ A] [ A] [ A] [ A]
1+
K1 K 2 K 3 K 4
4

We can simplify this by simply replacing the product K1 K 2 K 3 K 4 by a constant, K .

[ A]4
4
K
=
[ A]4
1+
K

(1.30)

This can be made general for a system with N infinitely cooperative sites in the
following equation.
[ A]N
K
=
[ A]N
1+
K
N

(1.31)

Equation (1.31) is called the Hill equation and is the inspiration for a frequently used
method of graphically displaying binding data in which there are cooperative sites (see
the next section). Equation (1.31) can be re-written in the following ways in terms of the

( N).

fraction of sites occupied, =

26

[ A]N
= K N
[ A]
1+
K

or

[ A]N
K

(1.32)

From the definition of K = K1 K 2 K3 K 4 etc. in equation (1.32), the midpoint of the binding
isotherm, at which = 0.5 is where [ A] = K 1/ N = ( K1 K 2 K 3 K 4 )1/ N , which is the geometric
mean of the multiple binding sites.
It is interesting to compare the binding isotherm for a system of infinite
cooperativity to the binding isotherm when there is no cooperativity, which is identical to
equation (1.32) setting N = 1 (see Figure 10.13).

27

Figure 10.13: A comparison of the forms of the binding isotherms for a protein with
N equivalent sites that are (A) independent, non-cooperative, or (B) coupled by
infinite positive cooperativity. The steepness of the binding isotherm can distinguish
these cases.

The main point of interest is that the steepness of the binding isotherm is not
made infinite by infinite positive cooperativity, but is limited by the number of
interacting sites. Consider the ratio of free ligand concentrations required to go from
10% saturation ( = 0.1 ) to 90% saturation ( = 0.9 ) of the binding sites.

[ A]N =
K
1

[ A]
[ A]

N
= 0.9
N
= 0.1

0.9
(1 0.9)
=
= 81
0.1
(1 0.1)

(1.33)

[ A] =0.9
= (81)1/ N
[ A] =0.1
The width of the binding isotherm is independent of the affinity ( K ) but depends on the
number of interacting binding sites. For simple, non-cooperative binding, the width of
the binding isotherm specifies that the concentration of ligand required to saturate 90% of
the sites is 81-times the concentration required to saturate 10% of the sites. We can
convert to a log scale of the free ligand to yield the following.
log

[ A] =0.9
= log(81)1/ N
[ A] =0.1
(1.34)

or
log[ A] =0.9 log[ A] =0.1 =

1.91
N

28

For all pH titrations of independent (non-cooperative) protonatable sites, N = 1 in

equation (1.34), and the width of a pH titration is 1.91 pH units, or about 2 pH units to go
from 10% to 90% protonated.
If we have multiple, infinitely cooperative sites, the width of the binding isotherm
is much less.

N =1

[ A] =0.9
= 81
[ A] =0.1

N =2

[ A] =0.9
= 811/2 = 9
[ A] =0.1

N =4

[ A] =0.9
= 811/4 = 3
[ A] =0.1

(1.35)

If one finds experimentally a binding isotherm in which [ A] = 0.9 [ A] = 0.1 < 9 , this can
only be interpreted to mean that there must be more than 2 coupled binding sites. Plots of
equation (1.32) are shown in Figure 10.14 with N = 1, 2 and 4 .

29

Figure 10.14: The binding isotherms of infinitely cooperative binding for a system
with 1 site (non-cooperative), 2 sites and 4 sites. These are graphs of equation (1.32),
[ A]N
N
K N = 1, 2 and 4 ,
with the values of K = 106 , 1012 and 1024 for =
[ A]N
1+
K
respectively, in order for the plots to cross where = 0.5 . The geometric mean of
the values of the intrinsic dissociation constants in each case is, therefore,
maintained at 10 6 M .

10.9 Graphically displaying binding data

There are a number of conventions for plotting binding isotherms, and different
disciplines in biology favor particular types of graphs for historical reasons and tradition.
These are summarized in Figure 10.15. We have already seen the Direct Plot and the
Log Plot as ways of displaying data. The latter is sometimes called a Bjerrum Plot, and

derives from the traditional way of plotting the protonation state of a weak base as a
function of pH. The Log Plot is the most useful way to display binding isotherms
because it allows the entire isotherm to be displayed in a way that makes it obviously if
saturation has been reached and, from the width, whether the system is exhibiting
cooperativity (e.g., Figure 10.15).

30

Figure 10.15: The origin and forms of the various plots used to display binding
isotherms.

The Reciprocal Plot and Scatchard Plot are devised so that simple binding
situations yield a straight line, and the slope and intercept of the lines give one the
binding stoichiometry and binding constant. For a situation with N independent and
equivalent sites, these are satisfactory to obtain the binding parameters. As we will
demonstrate in the next section, however, each of these graphing methods can be very
misleading if the binding is not as simple.
Finally, the Hill Plot is derived from the form of the Hill equation for N infinitely
positive cooperative sites. Real systems never display infinite coupling, but the form of
the equation yields a parameter called the Hill coefficient which is very frequently used

31

to quantify the extent of thermodynamic coupling. This will be shown in the next
section.
10.10 Example: The cooperative dimer

In this section we will pick up the example of the cooperative dimer, introduced
in Section 10.5 (see Figures 10.6 and 10.7). We will use this example to view the
various ways of graphing binding data.
The binding isotherm can be written as follows.

[ PA] + 2[ PA2 ]
[ P] + [ PA] + [ PA2 ]
(1.36)

[ A]
[ A] [ A]
+2
K1
K1 K1
=
[ A] [ A] [ A]
1+ 2
+
K1 K1 K1
2

or

[ A] [ A] [ A]
+
K1 K1 K1

= =
2 1 + 2 [ A] + [ A] [ A]
K1 K1 K1

Note that when = 1 , this equation yields the same binding isotherm as one obtains for 2
independent and equivalent sites (Henderson-Hasselbach form). Figure 10.16 shows
what happens when the positive cooperativity is increased, which is accomplished by
decreasing the value of the cooperativity parameter, . The curves shift progressively to
lower concentrations as decreases, which reflects the increased affinity for binding the
second ligand (decreasing dissociation constant). The midpoint, i.e., the concentration at
which half the sites are filled, is given by

[ A] =0.5 = ( K1 K 2 )1/2 = ( K1 K1 )1/2 = K1

(1.37)

which is the geometric mean of the two binding constants. In addition, the curves become
steeper.

32

Figure 10.16: Plot of equation (1.36) with three values of the cooperativity
parameter, , assuming the intrinsic dissociation constant for the first ligand is
K1 = 10 6 M . Smaller values of represent increasing positive cooperativity. The
midpoint of the binding isotherms go to lower concentrations and the curves become
more steep as gets smaller.

When << 1 , the equation simplifies to

[ A]2
K12
=
[ A]2
1+
K12
2

(1.38)

Equation (1.38) has the same form as the Hill equation, as it should since a very small
value for means very strong positive cooperativity. We can rearrange the terms in
equation (1.38) to yield the form of the Hill equation which forms the basis of the Hill
Plot.

33

[ A]2
=
1 K12

where =

(1.39)

The Hill Plot of (1.39) will yield a straight line. Note that the slope of this line, plotting

log
vs log[ A] is 2, the number of binding sites that are coupled.
1

However, this is not a realistic situation, since usually the value of does not justify
simplifying (1.36) to (1.39). For this reason, the Hill coefficient was devised.

The Hill coefficient: The Hill coefficient, nH , is the slope of the plot of log
vs
1

=0.
log[ A] at = 0.5 , equivalent to log

1 =0.5

d log

1 =0.5
nH =
d log[ A]

(1.40)

Figure 10.17 shows the Hill plots for the cooperative dimer ( K1 = 10 5 M ) with several

values of . The plots appear linear close to the midpoints but at the limits of very high
or very low concentrations, the slopes approach 1. Generally, one can obtain
experimental data only within one order of magnitude from the midpoint, so often the
Hill plot appears linear.

34

Figure 10.17: Hill plots for a cooperative dimer (equation (1.36) with K1 = 10 5 M
and = 1 (non-cooperative), = 0.1 and = 0.01 , where K 2 = K1 . The Hill
coefficient is the slope of the traces at the indicated points, where = 0.5 . The
values of the Hill coefficients are 1, 1.5 and 1.8 for = 1.0, 0.1 and 0.01 , respectively.

For non-cooperative binding, nH = 1 . For the cooperative dimer, the limit of the Hill
coefficient is nH = 2 for infinite positive cooperativity. We can determine an analytical
form for the Hill coefficient by taking the appropriate derivative of (1.36). The result is
nH =

2
1+

(1.41)

Equation (1.41) has the expected limits of nH = 1 for = 1 and nH = 2 for << 1 . The
dependence of the Hill coefficient on is shown in Figure 10.18.

35

Figure 10.18: Dependence of the Hill coefficient on the cooperativity parameter for
a cooperative dimer.
o
= RT ln , we can
The maximal cooperativity is at very low values of . Since Gcoupling
o
. This is shown in Figure 10.19.
also see the dependence of nH on Gcoupling

Figure 10.19: The dependence of the Hill coefficient on the coupling free energy for
a cooperative dimer.

36

Both positive and negative cooperativity are observed in biochemical systems, and we
can get a feeling for the appearance of the binding isotherms by comparing several
representative cases. We will compare three cases.
i) Non-cooperative, with K1 = K 2 = 10 4 M ( = 1 ).
ii) Positive cooperativity, with K1 = 10 4 M and K 2 = 0.1K1 ( = 0.1 ).
iii) Negative cooperativity, with K1 = 10 4 M and K 2 = 100 K1 ( = 100 ).
Note that there is no way just based on thermodynamics to distinguish between two
equivalent sites exhibiting negative cooperativity from two sites which are not identical
but which differ in binding affinity. The form of the binding isotherms are identical. In
order to distinguish, one would need additional information. For example, measuring the
off-rate constant from one site under conditions in which the first site is occupied and
under conditions when the first site is empty should distinguish the two cases. If there is
negative cooperativity, it is likely that the off-rate constant will be faster when the first
site is occupied than when it is empty, corresponding to the weaker affinity of the second
site. If there is no coopertivity, occupancy of other sites will have no influence.
We will now plot simulated data for these three cases, using equation (1.36) and
using the various ways to graph binding isotherms shown in Figure 10.15. The Log
Plots (or Bjerrum Plots) for the three cases are shown in Figure 10.20. For the example
of negative cooperativity, this way of graphing the data makes it clear that the saturation
is not reached, even at the highest concentrations of free ligand concentration. This gives
the appearance of a greater width for the binding isotherm than one can explain based on
the model of independent, equivalent sites ( = 1 ).

37

Figure 10.20: Log Plot for a cooperative dimer in which K1 = 10 4 M and

= 1, 0.1 or 100 .

The same data are plotted using the format of the Scatchard Plot in Figure 10.21.

Figure 10.21: A Scatchard Plot of the same data as in Figure10.20.

The concave and convex shapes are diagnostic of negative cooperativity (or sites with
different affinities) and positive cooperativity, respectively. However, because the
ordinate is the reciprocal of the free ligand concentration, the presentation is distorted,

38

and not so clearly comprehended by appearance. For the case of negative cooperativity,
the existence of the second, weak binding, site might be missed, for example.
Figure 10.22 shows a reciprocal plot of the same simulated data.

Figure 10.22: Recipocal plot of the same data as in Figures 10.20 and 10.21.

Note that all the information about the second binding site for the example of negative
cooperativity is crammed near the origin and could easily be missed. Also, the curvature
for the case of positive cooperativity might also be missed. A reciprocal plot is often used
precisely in those situations in which one wants to know the number of binding sites by
extrapolating to infinite free ligand concentration (or

1
= 0 ). But unless one knows
[ A]

whether it is appropriate to use a straight line to extrapolate, one could easily get an
incorrect answer.
10.11 The distribution of protein species in solution

Up to this point we have been concerned with measuring the average binding of a
ligand to a protein, but for systems with more than two states of the protein (site-occupied
or site-empty), the actually distribution of ligands is often very relevant. For example,
39

An oligomeric ligand-activated channel may only be opened when all the

binding sites are occupied by ligand.
A signal transduction pathway may be initiated only when more than one
receptor in a cluster is bound to the activating ligand.
Inhibitors and antagonists will complete with activators for a number of
processes, and only those species bound to the proper activator is biochemically active.
The protocol that we used to derive the expressions for the binding isotherm
(section 10.4) can also be used to write down the appropriate expressions to obtain the
fraction of protein in various possible states. We can continue with our discussion of the
cooperative dimer from the last section to illustrate this.
1. How much protein has no ligands bound as we vary the concentration of free
ligand? We approach this by writing the expression in terms of the protein species.
[ P]
[ P]
=
[ P ]total [ P ] + [ PA] + [ PA2 ]

(1.42)

Now, substitute as we discussed in section 10.4, remembering to insert the coefficients

accounting for equivalent species.
[ P]
1
=
[ P ]total 1 + 2 [ A] + [ A] [ A]
K1 K1 K1

(1.43)

Once we assign values to K1 and , this function will tell us the fraction of protein with
no ligand bound as a function of [ A] .
2. What fraction of protein has one ligand bound? We can use the same protocol.
[ PA]
[ PA]
=
[ P ]total [ P ] + [ PA] + [ PA2 ]

(1.44)

and substitute to get

40

[ A]
K1
[ PA]
=
[
]
[ A] [ A]
A
[ P]total 1 + 2
+
K1 K1 K1
2

(1.45)

3. What fraction of protein is fully saturated, with both sites full? Again, use the
same procedure.
[ PA2 ]
[ PA2 ]
=
[ P ]total [ P ] + [ PA] + [ PA2 ]

(1.46)

[ A] [ A]
[ PA2 ]
K1 K1
=
[ P]total 1 + 2 [ A] + [ A] [ A]
K1 K1 K1

(1.47)

and then

We can plot these functions to get a sense of what they can tell us. Lets assign a value
of K1 = 10 3 M and first look at the non-cooperative example with = 1 . This is shown
in Figure 10.23. At the concentration at which [ A] = K1 , the protein population is 25%
P, 50% PA and 25% PA2.

41

Figure 10.23: Distribution of protein species with no ligand bound (P), one ligand
bound (PA) or two ligands bound (PA2) for a dimeric protein with two identical
binding sites for ligand A, with K1 = K 2 = 10 3 M ( = 1 ).
Figure 10.24 shows the same situation, except in this case we set = 0.01 , so the

second dissociation constant is 100-fold less than the first. As a result, most of the
species in which one ligand is bound will also bind a ligand at the second site. Very little
of the species PA is present under any solution conditions.

Figure 10.24: Distribution of protein species for a dimeric protein with identical
sites but exhibiting positive cooperativity. K1 = 10 3 M and = 0.01 .
Figure 10.25 shows the protein distribution for the case of negative cooperativity,

where the second binding constant is 100-fold weaker (larger dissociation constant;

= 100 ). In this case, the binding of the second ligand is barely apparent over the range
of concentrations shown, which is a concentration range about as high as one might
employ in a biochemical study of ligand binding. A factor of 100-fold change in the
o
11kJ / mole or 2.7kcal / mole . In this
dissociation constant is equivalent to Gcoupling

42

case, the functional result is that the second site will remain unbound under reasonable
physiological conditions for most any ligand.

Figure 10.25: Distribution of protein species for a dimer exhibiting negative

cooperativity, with K1 = 10 3 M and = 100 . The thermodynamics is identical to
the situation in which the protein has two binding sites with different dissociation
constants K1 = 10 3 M and K 2 = 101 M .
10.12 The magnitudes and origins of coupling free energies: models of allostery

Coupling free energies between ligand binding sites have been quantified in
relatively few cases, but their magnitudes are of the order of
o
Gcoupling
1 to 3kcal / mole ( 4 to 12 kJ / mole ). This is sufficient to shift a

dissociation constant by 1 to 2 orders of magnitude (10- to 100-fold), which is significant

under physiological conditions. For comparison, consider that a single hydrogen bond
o
might contribute -20 kJ to the binding free energy ( Gbinding
). It only requires very small

structural or dyanamic changes to alter the net binding free energy by 4 kJ/mole. As a

43

rule, these sites are not adjacent, so the communication between sites is through the
protein (or macromolecule for the more general case).
10.12.1 Models of allostery
MWC model: The traditional models of allostery are based on conformational

changes induced in the protein by ligand binding. The MWC (Monod-WymanChangeux) model (2) has been quite successful and is still widely used. The model was

proposed to explain positive cooperativity in multisubunit proteins, and postulates that

the protein exists in two discrete quaternary states, R and T (for relaxed and tense)
that are in equilibrium. The quaternary structure is determined by the interactions
between subunits at the subunit interfaces, and these interactions are linked to ligandinduced changes at the ligand binding site. Ligand binding shifts the equilibrium from T
to R and, therefore, alters the observed properties of the protein. The ligand affinity of a
subunit is proposed to be determined by the tertiary structure of that subunit, but in the
MWC model, the tertiary structure of the subunits is coupled to the quaternary structure.
The quaternary structures are traditionally designated as either R or T. The ad hoc
assumption that there are only two states of the protein means that if, for example, one
has a tetramer with four identical sites, then either all the sites have a low affinity for the
ligand or all the sites have high affinity for the ligand within a given oligomeric protein.
In the MWC model, the quaternary structure is coupled to the tertiary structure of each
subunit, so a change of quaternary of the subunits changes the tertiary structure of all the
subunits, providing the explanation for the difference in ligand affinities. The
conformational coupling induced by ligand binding is proposed to extend to the subunit

44

interface but not into neighboring subunits. Coupling between binding sites is, therefore,
always mediated in the MWC model by changes in quaternary structure.
There are a number of examples where X-ray crystallography has clearly shown
allosteric proteins, such as hemoglobin (3, 4), to exist in distinct quaternary
conformations when ligands are bound compared to the unliganded protein. As a first
approximation, the MWC model has been extremely useful and durable.
KNF model: The second traditional model for allostery was initially proposed by

Linus Pauling and then elaborated Koshland et al (5), and now known as the KoshlandNmethy-Filmer (KNF) model. In this model, ligand binding results in an induced

conformational change (induced fit) which can alter binding sites elsewhere in the
protein. Ligand-induced conformational changes extend from one binding site to
another, independent of changes in quaternary structure. Sequential binding of ligands
can lead to a sequence of conformational changes, resulting in a number of distinct ligand
binding states of the protein. There is no constraint to two quaternary structures, as
proposed by the MWC model.
There are also many models devised for particular allosteric proteins that are
somewhere in between the MWC and KNF models in the sense that some constraints as
to the allowed ligand binding states are invoked (e.g., see (3) for a discussion of several
models to explain the ligand binding properties of hemoglobin).
For any particular system, the question is which thermodynamic model explains
the experimental data with the fewest adjustable parameters (intrinsic dissociation
constants, coupling free energies and stoichiometries). In other words, which is the

45

simplest model? Clearly, the MWC model, being a 2-state model, has the fewest
adjustable parameters, but for many systems it is not applicable.
Current views: As the experimental and computational tools have improved, the

description of proteins has evolved to include an important component of dynamics, a

constant motion between a collection or ensemble of states at equilibrium and with
similar chemical potentials. We have already seen in the examples of ligand binding in
the last chapter (sections 9.7 and 9.8) that the configurational entropy of the protein
appears to play a significant role in determining the thermodynamic driving force for
ligand binding. The same is true for the source of coupling free energy between binding
o
sites on a protein. The values of Gcoupling
are small, and, being a thermodynamic
o
parameter, Gcoupling
can originate anywhere in the system or be due to small changes
o
throughout the system. The origins of Gcoupling
might come from changes in hydrogen

bonds or electrostatic interactions between the ligand and protein, or might be due to
changes in the protein configurational entropy induced by ligand binding.
Once a thermodynamic model is devised to encompass the observed allosteric
changes in binding affinities, assigning specific coupling free energies between binding
sites, then the challenge is to understand the mechanism behind the coupling free energy.
This is part of the current challenge.
Several issues that are related to allostery are worth reviewing.
i) Lock-and-key, induced fit and conformational selection: The
discussion of allostery has historically been intertwined with discussions about the
relationship between protein conformation and ligand binding. The lock-and-key view
is that the protein pre-exists in the configuration that is an exact fit for the ligand. The

46

induced fit view is that the ligand binds and then induces the protein to alter its
conformation. Proteins in their native state are now known to exist as a large collection
or ensemble of states, each molecule visiting different conformations constantly through
global and local vibrational motions. Among these states are those with a high affinity
for the ligand, and states with little or no affinity for the ligand. If the predominant state
has a high affinity for the ligand, it would appear as a lock-and-key system, whereas if
the high affinity state is less favored, then ligand binding to this state would stabilize the
state, making it predominant. This conformational selection or population shift is not
the same as induced fit, but shares the feature that the protein conformation in the
presence of the ligand is different from the conformation prior to ligand formation (6-9).
(see Figure 10. 26).

Figure 10.26: Schematic illustration of an energy landscape of a protein.

The axis represents a generalized structural coordinate, so each location on the
curves represent distinct configurations of the protein. The ordinate is the standard
state free energy. Numerous local minima are indicated by the dips in each curve.
One predominant configuration is favored when the protein is not bound to the
ligand whereas a different configuration predominates when the ligand is bound.
Both configurations are always present at equilibrium, but their relative amounts
shift due to ligand binding. From (9).

47

However, recall that a small difference in the standard state free energy between
two equilibrated states can mean that one state might predominate by 10-fold or 100-fold
over another state. The state that is in the minority might be unobservable
experimentally, so a population shift might appear as induced fit.
True induced fit might be observed experimentally by observing the kinetics of
binding (see Chapter 8), in which case one might expect the rate of binding to reflect at
least two steps: initial complex formation, followed by a change in the protein (the
induced fit) leading to the final complex that is observed at equilibrium.
ii) A conserved pathway of residues linking cooperative sites: In systems where
there are large conformational changes that correlate with allostery, specific sets of
interactions between residues can be identified as being altered, for example, by moving
a helix due to direct connections with the ligand binding site. The pathway for conveying
the conformational changes in this manner is essentially a mechanical description. There
are also examples of allostery in which ligand-induced conformational changes have not
been observed, and in these cases, the evidence for a particular pathway through the
protein to convey allosteric information is more circumstantial (10-12).
iii) Ligand-induced conformational changes: At this point it seems that the large
majority of allosteric systems do involve observable ligand-induced conformational
changes in the protein. However, allosteric interactions need not involve conformational
changes, but can be entirely due to changes in dynamics, or the range of motions of the
protein being influenced by ligand binding (7-9, 13). The result of this is that the
coupling free energy will be largely entropic, tied to changes in protein configurational
entropy. This is schematically illustrated in Figure 10.27.

48

Figure 10.27: Protein energy landscapes illustrating how ligand binding can
either increase or decrease the number of protein configurations. On the left, the
free energy well is broad in the absence of the ligand, but is more narrow in the
presence of the ligand. This will result in a decrease in protein configurational
entropy. On the right, the opposite is observed. Ligand binding leads to a broader
bottom to the energy well, and more protein configurations are compatible in the
presence of the ligand than in its absence. From (9).

iv) Ligand-induced changes at protein-protein interfaces: Many allosteric

proteins are oligomeric, containing multiple subunits, with coupled sites located on
different subunits (or different domains of a single subunit). This was the inspiration for
the MWC model, which has proved to be very insightful. In many cases, it has been
experimentally demonstrated that ligand binding results in changes in the interactions
between the subunits. This can not only be seen in X-ray structures but also is evidenced
by changes in subunit dissociation constants coupled to ligand binding. Hemoglobin is
one example of this (4).
It is a natural extension of these observations to consider that interface regions
may be of general importance in conveying allosteric information between binding sites

49

in different subunits or domains, an essentially element of the MWC model. The

interface is a shared region that can communicate with the different binding sites (Figure
10.28). Changes induced in either the conformation or dynamics of residues at interface

regions could be critical to connecting different sites sharing that interface.

Figure 10.28: One possible origin of coupling free energy is due to alterations at the
subunit interfaces. Once one ligand binds, the interface is altered in some way, and
this can influence the binding free energy of ligands to equivalent (or different) sites.

The same concept would also apply to signal transduction pathways where ligand
binding alters protein-protein or protein-DNA interactions. When one speaks of a protein
being recruited to a task within the cell, this frequently is a result of allostery, where
the end result is in increased affinity of one protein for another.
We will now look at two examples that contrast in their explanations for the
observed allostery. Both involve homotropic cooperativity. Hemoglobin is reasonably
well by models derived from the MWC model. cAMP binding to the catabolite activator
protein, however, requires a different description.

50

10.13 Example: hemoglobin (3, 4)

The most intensively studied allosteric protein is hemoglobin, which exhibits both
homotropic and heterotropic cooperativity. The physiological function of hemoglobin is
to carry oxygen from the lungs to all the tissues in the body. Hemoglobin is a tetrameric
protein containing two subunits and two subunits, which are very similar. Each
subunit contains a heme, and the heme Fe binds to O2, forming a stable complex. The
four binding sites for O2 are considered to be identical.
Figure 10.29 shows the binding isotherm of hemoglobin plotted as Direct Plot

and also in the form of a Hill Plot. Also shown are the isotherms for the protein in the
low affinity T form and in the high affinity R form, as analyzed by the MWC model.

Figure 10.29: The experimentally observed binding isotherm for O2 binding to

hemoglobin presented as a Direct Plot (top) and Hill Plot (bottom). Positive
cooperativity results in the sigmoid shape of the Direct Plot, as opposed to the
hyperbolic shape observed for non-cooperative systems. Oxygen concentration is
expressed in terms of partial pressure (torr). Also shown are the calculated binding
isotherms for the R and T forms that can be used to model cooperative binding of
O2. The Hill coefficient for O2 binding to Hemoglobin is nH 3 .

51

Analysis using Adairs equation (1.4) yields macroscopic (statistical) binding constants.
Table 10.1 shows that the last oxygen to bind has a substantially higher affinity than does

the first oxygen. The binding affinity increases progressively as O2 binds.

Table 10.1: Comparison of dissociation constants for hemoglobin, subunits and
related compounds.
Macroscopic
dissociation constants
(mm Hg)
st
Hemoglobin: 1 O2 26
Hemoglobin: 4th O2 0.17
Isolated
0.52
Isolated
0.29
Myoglobin
0.51
Model Cpds
0.49

The binding free energies for the four consecutive (statistical) binding constants are given
in Table 10.2.
Table 10.2: Binding free energies corresponding to the four statistical binding
constants for O2 binding to hemoglobin.
-5.5 kcal/mole
GIo
GIIo

-6.2 kcal/mole

o
GIII

-6.6 kcal/mole

GIVo

-9.0 kcal/mole

Homotropic cooperativity and the MWC model:

The binding isotherm can be successfully modeled using the MWC model. A free
energy diagram illustrating the MWC model is shown in Figure 10.30. The protein is in
only two states, R and T. Each conformation of the protein has four identical binding
sites, with the R form having a much greater affinity than the T form. Cooperativity
results from mass action, shifting the population of the protein from the T form to the R
form as more O2 binds.
52

Figure 10.30: Free energy diagram of the MWC model of a tetrameric protein
binding ligand A, which is O2 for hemoglobin.

There are three adjustable parameters in the MWC model. It is the traditional to express
the MWC parameters in terms of association constants and not, as elsewhere in this text,
as dissociation constants.
i) The equilibrium constant for between the unliganded T and R species.
L=

[T ]
[ R]

G oL = RT ln L

(1.48)

ii) The intrinsic association constant for the ligand to the R form.

53

KR =

[ RA3 ]
[ RA2 ]
[ RA4 ]
[ RA]
=
=
=
[ R][ A] [ RA][ A] [ RA2 ][ A] [ RA3 ][ A]
(1.49)

GRo = RT ln K R
iii) The intrinsic association constant for the ligand to the T form.
KT =

[TA3 ]
[TA2 ]
[TA4 ]
[TA]
=
=
=
[T ][ A] [TA][ A] [TA2 ][ A] [TA3 ][ A]
(1.50)

GTo = RT ln KT
Figure 10.31 is another way to show the thermodynamic relationships between the 10

different states of hemoglobin in the MWC model.

Figure 10.31: Thermodynamic relationships between the 5 T species and 5 R species

postulated to be in solution in the MWC model of hemoglobin. Filled circles and
squares represent O2 bound at that site. All sites are considered to be equivalent.
KT and KR are intrinsic association constants. The macroscopic binding constants
are what one would obtain using a model-independent measurement if one were
measuring binding to the R species alone or the T species alone (e.g., K IT = 4 KT ,
etc.).

54

The fourth parameter in Figure 10.31 is the term c , which expresses the shift in the
equilibrium from the R to the T forms as more ligands are bound.
cL K 2 =

One can show that c =

[TO2 ]
[ RO2 ]

(1.51)

KT
by constructing a thermodynamic cycle (Figure 10.32).
KR

Figure 10.32: A thermodynamic cycle used to obtain the expression for K2, the
equilibrium constant for the single-liganded forms of T and R. Note that because of
the definition of the equilibrium constant, L, G3o = GLo = RT ln L , and
G3o = RT ln K 2 .

From Figure 10.32, one sees that

G1o + G2o = G3o + G4o

(1.52)

Therefore,

55

K1

1
1
K4
=
K 2 K3

K2 =
c=

(K2 =

[TO2 ]
[T ]
and K 2 L =
)
[ RO2 ]
[ R]
(1.53)

K 3 K1 KT
L = cL
=
K4
KR

KT
KR

The best fit of the MWC model for hemoglobin yields the following parameters:

L=

[T ]
= 107
[ R]

KT = 0.01 torr 1 or dissociation constant

1
= 100 torr
KT

K R = 10 torr 1

1
= 0.1 torr
KR

or dissociation constant

The intrinsic association constant for O2 increases by 1000-fold between the low affinity
T form and the high affinity R form of the hemoglobin binding sites.
Heterotropic cooperativity and the MWC model:

Several ligands act as negative allosteric effectors of O2 binding to hemoglobin.

Most notable are 2,3 diphosphoglycerate (DPG) and protons. Proton binding to
hemoglobin, for example, results in a lower affinity of O2. This is physiologically
important since it couples O2 binding to the CO2 content in the blood. In tissues, CO2 is
released as an end product of metabolism. This combines with water to for bicarbonate
and protons, thus acidifying the blood. The more acid pH lowers the affinity of
hemoglobin for O2, resulting in further release of O2 into the tissues. The opposite occurs
as the blood goes through the lungs. CO2 is exhaled, resulting in lowering the
bicarbonate concentration in the blood, so the pH goes up (more alkaline). The affinity

56

of hemoglobin for O2 increases, so more O2 binds to the protein. This latter effect is
known as the alkaline Bohr effect.
In the MWC model, all allosteric effectors influence hemoglobin by stabilizing
the T quaternary structure. This is pictured in the free energy diagram in Figure 10.33.

Figure 10:33: Modeling heterotropic cooperativity according to the MWC model of

hemoglobin. Ligand B binds (one per tetramer in the model), binds to the T form
with much greater affinity than the R form.

The intrinsic association constants are not altered, but the entire manifold of T forms of
the protein is stabilized (lower standard state chemical potential) with respect to the
manifold of R species. As a result, as O2 binds, the shift to the high affinity form of
hemoglobin is substantially less, lowering the affinity and also lowering the apparent
cooperativty.
57

Structural explanations of the observed thermodynamics:

When the X-ray structures of hemoglobin in the oxy and deoxy (or equivalent)
forms were determined, the structural explanation for the thermodynamics was
formulated by Max Perutz as the stereochemical model. The binding of O2 to the heme
Fe, causes the iron to move more into the plane of the heme. This displaces or pulls on
the histidine ligand binding to the opposite (distal) side of the heme, which, in turn,
results in displacement of the F -helix. This, in turn, displaces the FG corner, between
helices F and G, and involved in intersubunit contacts (see Figure 10.34). As a result,
intersubunit salt bridges are broken, destabilizing the T quaternary structure.

Figure 10.34: The structural elements which undergo a conformational change upon
O2 binding to a hemoglobin subunit. The FG corner is involved in intersubunit
contacts.

58

The hemoglobin tetramer can be considered to be a dimer of dimers. In the T

quaternary structure, corresponding to the deoxy form of hemoglobin, there are 8 salt
bridges, 2 per subunit, that help stabilize this structure. These are broken in the R
quaternary structure (the oxy form of hemoglobin). The result is that in the R form,
one of the dimers has rotated with respect to the other dimer, as shown in Figure
10.35. Ligand-induced subunit rotation is not unique to hemoglobin and is observed in

other allosteric proteins which can be analyzed in terms of R and T quaternary structures,
other examples being fructose 1,6 bisphosphatase (14) and phosphofructokinase (15).

Figure 10:35: The quaternary structure of hemoglobin is viewed as an 11 dimer

on top of a second 22 dimer. O2 binding results in rotation of the pairs.
Adapted from (3), originally from (16).

59

The connection between cooperativity and the interface between the pairs is further
demonstrated by the fact that dissociation of hemoglobin to form the dimer, results in
elimination of cooperativity and a large enhancement of the affinity for O2(17).
Two of the salt bridges contain an ionizable proton, and when these are broken,
protons are released. This is the structural cause of the alkaline Bohr effect. Protonation
stabilizes the deoxy or T form of hemoglobin.
Does the MWC model explain everything about hemoglobin cooperativity?

The MWC model makes predictions that have not borne out, and therefore
additional features are needed for a more complete theoretical framework for
understanding the thermodynamic linkages in hemoglobin. Basically, a large amount of
experimental evidence has shown that the subunits are not all in the same conformation in
the T state, nor in the R state. There are fixes that work well, but work continues. One
modification is to assume that there are two subunit conformations (t and r) that are in
equilibrium. The T state contains subunits mostly in the t configuration and the R form
has subunits mostly in the r configuration. (see (3)).
Major unanswered questions concern the actual origins of the coupling free
energies. In other words what does it mean when a structure is tense or strained.
Attention has focused on two regions: 1) the salt bridges at the intersubunit contacts; 2)
the core region near the heme. In the T quaternary structure, local interactions in these
regions are optimal or relaxed. Upon ligand binding, the conformational change driven
by the ligation makes these interactions less than optimal. Therefore, there is a free
energy cost to ligand binding when going from the going from the T to the liganded R
state. Once in the R state, this cost does not have to be paid so the apparent binding free

60

energy increases in magnitude. This is illustrated in the free energy diagram in Figure
10.36. The strain is a relatively small free energy change. If if

[T ]
= 107 then
[ R]

o
Gstrain
= 40kJ / mole (9.5kcal / mole) . This could come from many regions of the

protein.

10.14 Example: The catabolite activator protein from E. coli (18, 19)

In E. coli, the presence of sugars, such as lactose or arabinose, triggers processes

that increase the rate of transcription of genes that encode proteins required for the
utilization of the sugars, catabolic enzymes. The catabolite activator protein (CAP) is a
key element in this process. High sugar levels result in increased levels of cAMP, and
the cAMP binds to the catabolite activator protein. CAP is a homodimeric protein, and
each subunit has one cAMP binding site. CAP can exist in three different liganded states
in the presence of cAMP: CAP, (cAMP)CAP and (cAMP)2CAP (no ligand bound, one
ligand bound and two ligands bound). Upon forming the single-liganded form,
(cAMP)CAP binds to the operator region of DNA and recruits RNA polymerase, hence
inducing transcription of the appropriate genes. It is known that the form of the protein
with one ligand bound has a high affinity for DNA compared to the apo-CAP. Once
bound to the DNA, binding of the second cAMP is enhanced and the doubly-ligated
(cAMP)2CAP is the only form known from X-ray crystallographic studies.
This system exhibits multiple forms of allostery.
1. Homotropic negative cooperativity between the two cAMP binding sites.
2. Heterotropic positive cooperativity between the first cAMP binding and DNA
binding.
61

3. Heterotropic positive cooperativity between DNA binding and the second

cAMP binding.
Note that thermodynamics determines the binding affinities but not the rates or
sequence of reactions that occur. The rates will be controlled by concentrations and rate
constants. The sequence of events that has been proposed (18) is shown in Figure 10.37.

Figure 10.37: Structure of the homodimer catabolite activator protein (CAP)

showing bound cAMP in each site. A model of the sequence of events for the cAMPinduced binding of the catabolite activator protein to DNA. From (18).

We will review some of the work relating only to the issue of the origin of the coupling
free energy responsible for the negative homotropic cooperativity. In the absence of
DNA, the affinity of the second cAMP to bind to the protein is about 100-fold less than
the affinity of the first cAMP. The two binding sites are located 24 apart, so there is no

62

direct connection. Figure 10.38 shows the ITC determination of the thermodynamic
parameters for cAMP binding to CAP.

Figure 10.38: ITC determination of the binding parameters for cAMP to the
catabolite activator protein. Work was performed with the N-terminal fragment.
Adapted from (19).

The coupling free energy is easily determined from these data.

o
Gcoupling
= (G2o G1o ) = 7.5 (10.3) = +2.8kcal / mole

(1.54)

The ITC data show that the binding of the second cAMP has a less favorable binding free
energy ( G2o > G1o ) entirely due to a less favorable entropic contribution. The
difference in the enthalpic portion of the binding free energy actually favors a stronger
interaction for the second cAMP ( H 2o < H1o ).

63

A reasonable explanation for these results comes from dynamics experiments

which determine the extent of motions of the protein as well as from supportive
computational data. These results show the following.
1. Binding of the first cAMP results in a conformational change in the subunit
containing the binding site, but not second subunit. There are, however, changes in the
protein dynamics of both subunits, particularly an increase in the dynamics in the secmsec range. It is estimated that there is an overall configurational entropic penalty for
binding of the first cAMP of about T S1,oconfig 3.2kcal / mole .Hence, the strong
favorable T S1o very likely results from the release of bound solvent upon cAMP
binding since the contribution from the configurational entropy of the protein is likely to
be unfavorable.
2. Binding of the second cAMP has a dramatic affect on the dynamics of both
subunits, causing a large constraint of protein motions (fewer microscopic states can be
accessed, hence the entropy decreases). The effect is strongest at the interface between
the two subunits. Hence, there is expected to be a large, unfavorable change in the
configurational entropy of the protein upon binding the second molecule of cAMP. The
estimate is that the entropic penalty of binding the second cAMP is much larger than for
the first cAMP, S2,o config 15kcal / mole . Presumably, the displacement of solvent upon
binding the second cAMP is similar to the first cAMP, so the favorable component to the
change in entropy of binding from solvent will be about the same. The large unfavorable
change in protein dynamics upon binding the second cAMP compared to the first, results
in a lower affinity by 100-fold, corresponding to a coupling free energy of

+2.8kcal / mole .

64

3. The X-ray structure of the doubly ligated CAP shows that there is no
difference between the subunits. The interactions between each subunit and the cAMP
appears to be identical.
Figure 10.39 summarizes these results pictorially.

Figure 10:39: Schematic of the changes induced by the first and second cAMP
binding to the N-terminal fragment of the catabolite activator protein (CAP). From
(19).
10.16 Summary

Allostery plays a major role in biological functions, from enzymology to signal

transduction and assembly and sorting pathways. Often, explanations are expressed in
mechanical terms, largely driven by observed changes in structure by X-ray
crystallography. This is reasonable and provides a useful framework to understand the
principles, but the approach can be very limiting. Allostery is a thermodynamic process
and involves relatively small changes in the chemical potentials of the interacting species.
It is useful to keep this in mind. The mechanical, enthalpic view of allostery may be
sufficient and a good approximation in many cases. However, entropic considerations
based on molecular dynamics appear to play an important role. The thermodynamic
measurements provides the essential first step in characterizing the system. The
structural and dynamic origins of the measured H o and S o values provide insight into
the molecular mechanisms.

65

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