Lecture 8

Binding and kinetics

Antoine van Oijen
BCMP201 Spring 2008

Donald T. Hayne
Biological Thermodynamics
James Goodrich, Jennifer Kugel
Binding and Kinetics for Molecular Biologists

Goals

- Quantitative measurements of biological binding reactions

- Affinities
- Cooperativity in binding
- Kinetics

Practical use!!!

Assays: how much is bound?

Protein-protein, protein-DNA, protein-ligand,

Assays that separate complexes from a solution

- Filter-binding (or cell-binding)
- Gel-filtration chromatography
- Electrophoretic mobility shift assays (EMSAs/ gel-shift)
Assays that detect complexes in solution
- Fluorescence (quenching, anisotropy, FRET)
- Protection assays (Rnase, Dnase footprinting)
Assays in which a biomolecule is bound
- Affinity resins
- Surface plasmon resonance
(More details later in the semester)

Bimolecular interactions

Binding is not all-or-nothing:

A+B

kon
koff

AB

Portion of A and B will be bound, portion will be free

Equilibrium
Reaction is in equilibrium when concentrations do not change:
X

kon
koff

d[Y]
= [X ] " kon # [Y] " koff = 0
dt

(mass action law)

!
(unimolecular reaction)

Equilibrium
Binding (bimolecular reaction):

A+B

kon
koff

AB

Reaction is in equilibrium when concentrations do not change:

d[AB]
= [A] " [B] " kon # [AB] " koff = 0
dt

(mass action law)

Equilibrium is reached when:

[A] " [B] " kon = [AB] " koff

Equilibrium
! is still dynamic!!!

Equilibrium dissociation constant KD

Equilibrium is reached when:
[A] " [B] " kon = [AB] " koff

Rearrange to define equilibrium dissociation constant KD:

KD =

koff [A] " [B]

=
kon
[AB]

When [A]=Keq, 50% of B is bound to A

Units
Units:
KD =

[A] " [B]

[AB]

{M} =

{M} " {M}

{M}

(Conversely, equilibrium binding constant, KB, is defined as:

!

KB =

[AB] !
[A] " [B]

{M "1} =

{M}
{M} # {M}

Rate constants:

KD =

koff [A] " [B]

=
kon
[AB]

koff: {s-1}
kon: {M-1s-1 }

Where does this KD come from?

From Lecture 5:

How to measure KD ?
KD =

[A] " [B]

[AB]

Measure [A], [B], and [AB]?

Introducing [A]Total=[A]+[AB]:

[AB]
[B]
=
[A]Total KD + [B]

Figure from: Goodrich, Kugel

Experimental considerations
[A] constant; titrate B
Measure fraction bound

[AB]
[B]free
=
[A]Total Keq + [B]free

If [A]Total << KD, then [B][B]+[AB]

!

Figure from: Goodrich, Kugel

No need to measure [B],

Just take [B]Total!

Logarithmic versus linear display

Figure from: Goodrich, Kugel

As a corollary: Choose your titrations logarithmically!

1, 3, 10, 30, 100, 300 nM, or
2, 4, 8, 16, 30, 60, 180, 360 nM, instead of
50, 100, 150, 200, 250, 300 nM

Example: Repressor binding to DNA

DNA + R

kon
koff

DNA-R

KD10-10 M for operator DNA (specific binding)

KD10-4 M for non-operator DNA (non-specific binding)

In E. coli, how much repressor is bound non-specifically to DNA

and how much is free?
[non-operator DNA] 106 / 1 m3 10 mM (107 bp/genome; 10 bp/site; volume. E.coli 1 m3)
[R]
F=
=
[R] + [R " DNA]

[R]
[R]

[DNAnon ]
[R " DNA]

[DNAnon ]
[DNAnon ]
+ [R " DNA]
[R " DNA]
[R " DNA]

KD
10#4 M
= #4
= 0.01
KD + [DNAnon ] 10 M + 10 -2 M

Hardly any free repressor; almost all bound to nonspecific DNA!

Non-cooperative versus cooperative

Not Cooperative

000 + B

Protein

00B + B

00B
0BB

Cooperative

000 + B

Protein

00B + B

K
K

00B
0BB

can be positive or negative (positive or negative cooperativity)

Cooperative binding
Simplification:
A + nB

kon
koff

(perfect cooperativity)

ABn
KD =

[A] " [B]n

[ABn ]

Rearrange (next Problem Set???):

!
# Y &
log%
( = nH ) log[B] " log KD ,
$1"Y '
where Y=[ABn]/[A] total

Cooperative binding

# Y &
log%
( = nH ) log[B] " log KD ,
$1"Y '
where Y=[ABn]/[A] total
Figure from: Goodrich, Kugel

Hemoglobin

Reaction kinetics

++

Energy profile for a

generic chemical reaction:

Gibbs free energy (G0) determines ratio of reactants/products

(thermodynamic properties), activation energy (G++) determines
rates (kinetics)

Figure from: Haynie, Biological Thermodynamics

Equilibrium thermodynamics does not provide any

information on rates of chemical changes!

(dynamite versus nitroglycerin)

Rate of reaction
Reaction rate = a measure of how fast the concentration of reactants /
products changes with time
Example: hydrolysis of ATP into ADP

ATP

ADP + Pi

J ="

d[ATP]
d[ADP]
d[Pi ]
=+
=+
dt
dt
dt

Figure from: Haynie, Biological Thermodynamics

Reaction rate:

10

Rate constant and order of reaction

Reaction rate/velocity is related to concentration of reactant:

J = k[A]n
n is order of reaction (often identical to stoichiometry)
k is rate constant (dont confuse with binding constant)

!
We saw that J = "

d[A]
, so k will have:
dt

Per second (s-1) as unit for 1st order reaction,

Per molar per second (M-1s-1) as units for 2nd order reaction

1st order reaction

1st order reaction A
Combining J = "

"
!

d[A]
with J = k[A]
dt

d[A]
= k[A]
dt
!

!
[A]
= e("kt)
[A]0

1
d[A] = "kdt
[A]
ln[A] = ln[A]0 " kt

Figure from: Haynie, Biological Thermodynamics

Integrate ( " dx = ln x + C):

x
!

gives:

11

2nd order reaction

2nd order reaction 2A
Combining J = "

"
!

1
1
dx = # + C):
x
x2

1
d[A] = "kdt
[A]2
1
1
=
+ kt
[A] [A]0

Figure from: Haynie, Biological Thermodynamics

d[A]
with J = k[A]2 gives:
dt

d[A]
= k[A]2
dt
!

Integrate ("

[A]
1!
=
[A]0 1+ kt

1st and 2nd order reactions

1st order:

2nd order:

[A]
1
=
[A]0 1+ kt
Figure from: Haynie, Biological Thermodynamics

[A]
= e("kt)
[A]0

12

Half-times and rate constants

Half time t 1/2 is not the same as k-1 :

[A]
= 0.50 = e("kt1/ 2 )
[A]0

" ln 2 = "kt1/ 2

# t1/ 2 =

ln 2 0.693
$
k
k

Temperature effects
Rates depend on temperature

A+B

kon
koff

AB

Arrhenius: k = Ae(" #G

++

/ RT)

ln k = ln A " #G ++ / RT
!

13

Reversible reaction
kon

A+B

koff

AB
d
[A " B] = kon [A][B] # koff [A " B]
dt
Formation
(2nd order)

!
Under equilibrium,

d
[A " B]
dt

Dissociation
(1st order)

equals zero:

[A][B] koff
=
= KD
[A " B] kon

Relation between KD, kon/off, and G

++

* -G ++
*
G0= Goff++
on

A+B
AB

!
In terms of free energies:

Figure from: Haynie, Biological Thermodynamics

[A][B] koff
=
= KD
[A " B] kon

++

++
++
koff Ae("#Goff / RT)
("(#Goff
"#Gon
) / RT)
("#G 0 / RT)
KD =
=
=
e
=
e
++
kon Ae("#Gon / RT)

14

Rates of binding and dissociation

A+B

kon
koff

AB

Association rate for two objects with diffusion coefficients D1 and D2

and diameter r1 and r2:
kdiff=4NA(D1+D2)(r1+r2)

(units: {mol-1}{cm2s-1}{cm} = {M-1s-1}

For a small ligand and protein: kdiff 109 M-1s-1,

for two proteins: kdiff 106 - 107 M-1s-1
This rate can be further slowed down if a conformational change
needs to take place before binding

Example: Repressor binding to DNA

DNA + R

kon
koff

DNA-R

d
[R " DNA] = kon [R][DNA] # koff [R " DNA]
dt
Formation
(2nd order)

Dissociation
(1st order)

It takes 0.1 seconds to switch off gene expression in E.coli after

lactose depletion. What is kon?

d
[R " DNA] # kon [R][DNA]
dt
With ~10 repressors per E.coli and [DNA]10-9 M (1 operator sequence in 1 m3 cell),
kon needs to be at least 109 M-1s-1 (is actually measured to be 1010 M-1s-1)
!

How come this is much faster than diffusion limit???

15

1D sliding along DNA to speed up kon

BWH (Berg, Winter, von Hippel) model:
Combine 3D diffusion (hopping) with
1D diffusion (sliding).
Scan short stretch of DNA by 1D
search, then jump to different area.
Length explored by one 1D sliding event:

"L(# ) = D1D #

(1D random walk)

Typical duration will be =1/knonsp.off:

"L = D1D / knonsp.off

Remember, repressors spend 99% of time on nonspecific DNA:

!
L(t) = tknonsp.off D1D / knonsp.off
Total length explored L(t) is linear with time!

1D sliding: the numbers

D1D 10-9 cm2/s (limited by rotational drag)
knonsp.off 10 s-1
L() 100 nm (300 bp)
100 kb of DNA is searched by single
repressor in half a minute
Searching 100 kb with only 1D sliding would take
Ttotal = L2total/D1D 3 hours!

Now we understand why 99% of repressor is bound to nonspecific DNA:

Theyre actively involved in the search process.

16

Folding revisited: a riboswitch

Folded RNA that binds small molecule (aptamer)
Plays role in regulation of gene expression

How does it fold?

?
Single-molecule probing of RNA folding

Liphardt et al., Science (2001)

17

Pulling at an RNA hairpin

Force-extension curve of
single RNA unfolding/folding

Liphardt et al., Science (2001)

Force tilts free-energy diagrams

Along the reaction coordinate, an

amount of energy equal to force
times displacement is added

G = -Fx
G = G0 - Fx

18

Pulling at an RNA hairpin

$ #G0 " F#x '

$ #G '
P(unfolded)
= exp&"
)
) = exp&"
P(folded)
kT
% kT (
%
(
Liphardt et al., Science (2001)

Pulling at an RNA hairpin: kinetics

Single-molecule kinetics:
Direct observation of kopen and kclose

Liphardt et al., Science (2001)

19

Unfolding a riboswitch

Unfolding a riboswitch

20

Take-home message
Equilibrium constant K is related to free energy difference G0
between initial and final state, rates k are related to free energy
differences G between initial/final state and transition state
++

[A][B] koff
=
= KD
[A " B] kon

* -G ++
*
G0= Goff++
on

A+B
AB

++

++
++
k
Ae("#Goff / RT)
("(#Goff
"#Gon
) / RT)
("#G 0 / RT)
KD = off =
=
e
=
e
++
kon Ae("#Gon / RT)

21