Binding

A substance is not effective unless it is linked to another.’’

Paul Ehrlich
 The Role of Binding in the HIV
Life Cycle

One of the first

processes in the
infection process is
the binding of the
virus to the host cell.
Hemoglobin Binding Curves

``A substance is not effective unless it is

linked to another.’’ - Paul Ehrlich

O2 binding curves
Transcriptional Regulation
•  Regulation takes place very far upstream. In
particular, the “decision” is made whether or not
to produce mRNA.
•  Question: What are the molecules that mediate
this control?
•  Molecular binding events on DNA are a key
mechanism of control.
 Antibody-Antigen Binding
The Immune System

Molecular recognition mediated by binding reactions is at

the centre of immune response.
 An estimate of the contribution of the
various factors to ΔGo for binding of a ~
30 residue proteins (T = 300 oK).

A typical form in
which energy of
interactions between
Etot = ∑ qφel + Aφsteric + Bφdisp + Edesolv
two proteins or protein
and small molecule
Ionic pairs + Van der Waals removal of water
can be written H-bonding from the contact
Evaluation of binding
A + B ↔ AB
Thermodynamic properties
ΔH enthalpy change
ΔS entropy change
ΔC heat capacity change

Affinity – strength of binding

KA (affinity constant) or KD (dissociation constant)
KD=1/KA

Kinetics
kass or kon association rate constant or on rate
kdiss or koff dissociation rate constant or off rate

Mechanical properties (e.g. unbinding force, elasticity)

 KA
Affinity A + B → AB
It is measured with the standard free energy of binding ΔG0.
0 0 0
ΔG = ΔH − TΔS
ΔG0 < 0 – binding is favoured.
ΔGo is the standard state which assumes all components are at
the standard state concentration of 1 M (mol.L-1).
0
ΔG = − RT ln K A
Association constant KA [ AB] ΔGBA

K = =e
−
RT

[ A][ B]
A
q  The units (M-1)
q  The ratio of [products] versus [reactants] at equilibrium.
q  Higher affinity = higher KA
q  Depends on B concentration
Dissociation constant KD A + B ← AB
KD

q  Concentration of A at which half of B is

KD =
[ A][B ]
bound ([B]=[AB])
q  Units are M
[AB]
q  Higher affinity = lower KD

The complex lifetime – is independent of the concentration, it

depends only on the bond energy (KD is described by k-1).

Allosteric activators of enzymes e. g. NAD:

KD = 0.1 µM to 0.1 mM.

Antibody-antigen interaction
KD = 0.1 mM to 0.0001 pM.
 Types of binding simple

all binding sites are

equivalent and independent
cooperativity heterogeneity difficult
all binding sites are all binding sites are
equivalent and not independent independent but not equivalent

heterogeneity cooperativity very

all binding sites are difficult
not equivalent and not independent

Average number of ligand molecules bound to macromolecule:

concentration of A bound to B
n=
Total concentration of B n 0≤n ≤n
θ=
Fractional saturation n 0 ≤θ ≤1
Single binding site A + B ⇔ AB

0 ≤ n ≤1 n =θ

n=
[AB]
KD =
[A ][B]
⇒ [AB] =
[A ][B]
[B]+ [AB] [AB] KD

[A ][B]
n=
[AB]
=
K d

[B]+ [AB] [B]+ [AK][B] d

n=
[A]
K d + [A ]
‘titration curve’
(Langmuir isotherm)
KD can be obtain directly from
the binding curve.

θ=
[A] ⇒ θ = 1
K d + [A ] [A] K d + [A ]
1 1 1 1
= − + =
K d + [A ] K d K d K d + [A ]
1 K d + [A] − K d 1 [A] ⇒
− = −
Kd K d (K d + A) K d K d (K d + A)
Scatchard plot
θ 1 θ θ
versus θ
= − [A]
[A] K d K d
Straight line
Receptor occupancy is a hyperbolic function of [L ]
(Langmuir adsorption isotherm)

Bmax 1 Kd = 1
Kd = 3
[ L]
θ= 0.8
Kd = 10
K d + [ L] B1( x)
0.6
B2( x)
B3( x)

Kd has the dimension of 0.4

concentration and should be

measured in the same units 0.2

as L (M).
0
0 10 20 30 40 50

L
Note that for a shallow curve it is hard to say where it saturates
Binding to n identical and independent binding sites
Average number of ligands n[A ] n
bound per protein:
n = nθ = θ=
K d + [A ] n
•  Single ligand species.
•  All sites have the same Kd,. [A]
•  Sites are independent. θ=
•  Fractional saturation is identical. Κ d + [A ]
Determination of the number of sites
n n n Scatchard plot
0.14

= − 0.12
[A] Κ D Κ D 0.1

0.08

<n>/[I]
0.06
y = ax + b
n
0.04
1
a=− b= 0.02

0
Κ D
Κ D
0 0.5 1 1.5 2
<n>
The Hill coefficient – h, a measure of cooperativity
h
n[A ]
n= h
Κ d + [A ]
Hill plot
n n θ
θ= log = log = h log[A] − log Κ d
n−n 1−θ
n
Intercept at log{θ/(1- θ)} = 0
gives logKD.

Hill plots of
oxygen binding
for myoglobin
and hemoglobin.
Hemoglobin vs. Myoglobin
[ L]n
θn = n
K n + [ L]
1 Myoglobin, n = 1

0.8

B1( x) 0.6
Hemoglobin, n = 2.8
B3( x)

0.4

0.2

0
0 2 4 6 8 10

pO2 (kPa)
pO2 in tissues
Hill Plot
Slope at log{θ/(1- θ)} = 0 is the Hill coefficient, h.
nh = 1 for non-cooperative binding
nh < 1 for negative cooperativity
nh > 1 for positive cooperativity
nh = n (number of sites) for infinitely
positive cooperativity.
x
independent θ=
binding kd + x

cooperative xn
θ=
binding kd + x n

n – Hill coefficient