BEH.

420 Lecture III: Multivalent Binding Equilibrium page 1

Today would like to compare the effect of multiple (identical) interactions on the overall
binding of two molecules.

The basic paradigm is to compare:

Figure 1: Multivalent binding equilibrium.

Are G1 and G2 related? The simple guess is G2=2G1, but we will see that this is not
true.

There are many examples of multivalent binding interactions in biology and in the
experimental approaches used to analyze biological systems.

Figure 2: Examples of multivalent binding in biological systems.

General effect of multivalency: enhances affinity over single interaction (more

interactions; tethering produces high local concentration). The reader contains a
C&E News article that illustrates some applications of multivalent interactions.

Thinking about multivalency: Jencks, Proc. Natl. Acad. Sci. U.S.A. 78: 4046 (1981).
BEH.420 Lecture III: Multivalent Binding Equilibrium page 2

Figure 4: Multivalent binding free energy.

Figure 5: Multivalent binding free energy, involving unary reactions.

The Kus above refer to unimolecular dissociation constants, because the reactions are
intramolecular between units tethered together.

K d = K d K u = Ku K d
AB A B A B

[M] [M][1] [1][M]

Thermodynamic cycle: Total free energy change for binding has to be independent of
route.

G AB = G A + RT ln K u = G B + RT ln K u
B A

Figure 6: Defintion of Kd in terms of effective concentration.

B
Kd
[ B]eff = B
(units of M)
Ku
BEH.420 Lecture III: Multivalent Binding Equilibrium page 3

AB A B
We can measure: K d , Kd , Kd
AB AB
Kd K
= Ku = d B
B A
We can compute: K u A
,
Kd Kd
Then, we can compute: [A]eff , [B]eff (note: [A]eff = [B]eff )

B B A
Kd Kd Kd
[ B]eff = B
= AB
Ku Kd
A B A
K K K
[ A]eff = d A = d ABd
Ku Kd

Figure 7: Behavior of effective concentration.

Intuitive meaning of effective concentration:

1. [B]eff >> [AB], the binding of A positions B so that it is locally at a very high
concentration near the binding pocket and capable of binding.
2. [B]eff is the concentration of soluble B we would need in bulk solution to produce
the same (binding) effect (intermolecularly) achieved by AB binding
(intramolecularly).

Angry dog analogy: How many loose dogs would you need to put in the room to match
frequency of being bitten by a single dog chained to your leg? How does this number
change with the length of the chain?

Comments:
1. Lower limit of [B]eff is zero. E.g. short, rigid linker prevents intramolecular
binding.
2. Upper limit: no upper limit (not 55M the water concentration in aqueous
solution).

Figure 8: An example of high effective concentration.

BEH.420 Lecture III: Multivalent Binding Equilibrium page 4

The take-home message is that we can not predict

KdAB from KdA and KdB alone, so dont even try.

Receptor Cross-linking (see L&L 4.3)

Figure 9: Cartoon for receptor cross-linking.

dC1
= 2kon L0 ( P0 C1 2C2 ) koff C1 k xC1 ( P0 C1 2C2 ) + 2k xC2
dt
binding dissociation cross-linking de-cross-linking

dC 2
= k x C1 ( P0 C1 2C 2 ) 2k x C 2
dt

where kx, k-x are cross-linking and de-cross-linking dissociation constants

generally kx has units number/(cell time) and k-x has units 1/time.

dC1 dC 2
at equilibrium (steady state): = =0
dt dt
1 + 1 + 4 1 1 + 2 1 + 4
C1eq = P0 C 2eq = P0


2 2 2

L0
= = dimensionless ligand concentration
L0 + K d / 2
= (1 ) K x P0 = dimensionless cross-linking parameter
k
Kx = x
kx

Cross-linking can be functionally significant for receptor signaling (e.g., TCR) so the
concentration of cross-links (C2eq) as a function of ligand (L0) is important.
BEH.420 Lecture III: Multivalent Binding Equilibrium page 5

Figure 10: Bivalent ligand plot.

- symmetric with max at Kd/2 = L0

- number of cross-links increases with Kx and P0
- maximum number of cross-links = P0/2

The cross-linking model assumes that membrane proteins are mobile. With biosensors,
this may not be the case. Using Poisson statistics, we can calculate probability
that two proteins are close enough for bivalent interaction.

Simplified treatment of multivalent binding: [Muller et al., Analytical Biochemistry 261:

149 (1998).]
- antibody analyte, a sphere wherein 2 ligands could be bound
- Poisson statistics for distribution of immobilized ligand
- Conceptual partition of ligands into 2 types:
o Monovalent-capable Lm
o Bivalent-capable Lb
- Balance equations
P + Lm
PLm

P + Lb
PLb
PLb + Lb
PLb Lb
- Write down material balance (eqns 13-15 from paper)
- Describe experimental data reasonably well