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

1
 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)

2
 Bimolecular interactions

Binding is not all-or-nothing:

kon
A+B AB
koff

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

Equilibrium

Reaction is in equilibrium when concentrations do not change:

kon
X Y
koff

d[Y]
= [X ] " kon # [Y] " koff = 0 (mass action law)
dt

!
(unimolecular reaction)

3
 Equilibrium

Binding (bimolecular reaction):

kon
A+B AB
koff

Reaction is in equilibrium when concentrations do not change:

d[AB]
= [A] " [B] " kon # [AB] " koff = 0 (mass action law)
dt

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:

koff [A] " [B]

KD = =
kon [AB]

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

4
 Units

Units:
[A] " [B] {M} " {M}
KD = {M} =
[AB] {M}
(Conversely, equilibrium binding constant, KB, is defined as:

! [AB] ! {M}
KB = {M "1} = )
[A] " [B] {M} # {M}

! Rate constants:
!
koff: {s-1}
koff [A] " [B]
KD = =
kon [AB]
kon: {M-1·s-1 }

Where does this KD come from?

From Lecture 5:

5
 How to measure KD ?

[A] " [B]

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

[AB] [B]
! Introducing [A]Total=[A]+[AB]: =
[A]Total KD + [B]

Figure from: Goodrich, Kugel

!

Experimental considerations

• [A] constant; titrate B [AB] [B]free

• Measure fraction bound =
[A]Total Keq + [B]free
If [A]Total << KD, then [B]≈[B]+[AB]

!
No need to measure [B],
Figure from: Goodrich, Kugel

Just take [B]Total!

6
 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

kon
DNA + R koff
DNA-R
KD≈10-10 M for operator DNA (specific binding)
KD≈10-4 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 µm3 ≈ 10 mM (107 bp/genome; 10 bp/site; volume. E.coli 1 µm3)
[DNAnon ]
[R]
[R] [R " DNA] KD 10#4 M
F= = = = #4 = 0.01
[R] + [R " DNA] [DNAnon ] [DNAnon ] KD + [DNAnon ] 10 M + 10 -2 M
[R] + [R " DNA]
[R " DNA] [R " DNA]

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

!

7
 Non-cooperative versus cooperative

B Not Cooperative
B B K
000 + B 00B
Protein K
00B + B 0BB

B Cooperative
B B K
000 + B 00B
Protein τK
00B + B 0BB

τ can be positive or negative (positive or negative cooperativity)

Cooperative binding

Simplification:
kon
A + nB ABn (perfect cooperativity)
koff

[A] " [B]n

KD =
[ABn ]

Rearrange (next Problem Set???):

!
# Y &
log% ( = nH ) log[B] " log KD ,
$1"Y '

where Y=[ABn]/[A] total

8
 Cooperative binding

# Y &
log% ( = nH ) log[B] " log KD ,
$1"Y '

where Y=[ABn]/[A] total

Figure from: Goodrich, Kugel

!

Hemoglobin

9
 Reaction kinetics

Equilibrium thermodynamics does not provide any

information on rates of chemical changes!

Figure from: Haynie, Biological Thermodynamics

++

Energy profile for a

generic chemical reaction:

Gibbs free energy (ΔG0) determines ratio of reactants/products

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

(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
Figure from: Haynie, Biological Thermodynamics

Reaction rate:

d[ATP] d[ADP] d[Pi ]

J =" =+ =+
dt dt dt

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 (don’t confuse with binding constant)
!

d[A]
We saw that J = " , 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 P

d[A]
Combining J = " with J = k[A] gives:
dt

d[A] 1
" = k[A] d[A] = "kdt
dt ! [A]
!
1
Figure from: Haynie, Biological Thermodynamics

Integrate ( " dx = ln x + C): ln[A] = ln[A]0 " kt

x
! !

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

11
 2nd order reaction

2nd order reaction 2A P

d[A]
Combining J = " with J = k[A]2 gives:
dt
d[A] 1
" = k[A]2 d[A] = "kdt
dt ! [A]2
!
1 1 1 1

Figure from: Haynie, Biological Thermodynamics

Integrate (" dx = # + C): = + kt
! x2 x [A] [A]0
!

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

1st and 2nd order reactions

[A] [A] 1
1st order:
= e("kt) =
[A]0 2nd order: [A]0 1+ kt
Figure from: Haynie, Biological Thermodynamics

! !

12
 Half-times and rate constants

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

[A] ln 2 0.693
= 0.50 = e("kt1/ 2 ) # " ln 2 = "kt1/ 2 # t1/ 2 = $
[A]0 k k

Temperature effects

Rates depend on temperature

kon
A+B 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 Dissociation
(2nd order) (1st order)
!
d
Under equilibrium, [A " B] equals zero:
dt

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

Relation between KD, kon/off, and ΔG

Figure from: Haynie, Biological Thermodynamics

[A][B] koff
= = KD ΔG0= ΔGoff++
* -ΔG ++*
[A " B] kon ++ on

A+B

! AB

In terms of free energies:

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

14
 Rates of binding and dissociation

kon
A+B koff
AB

Association rate for two objects with diffusion coefficients D1 and D2

and diameter r1 and r2:

kdiff=4πNA(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

kon
DNA + R koff
DNA-R
d
[R " DNA] = kon [R][DNA] # koff [R " DNA]
dt

Formation Dissociation
(2nd order) (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 µm3 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 τ=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(τ) ≈ 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:

They’re 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

ΔG = -F·Δx

ΔG = ΔG0 - F·Δx

18
 Pulling at an RNA hairpin

P(unfolded) $ #G ' $ #G0 " F#x '

= 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 ΔG0

between initial and final state, rates k are related to free energy
differences ΔG‡ between initial/final state and transition state

ΔG0= ΔGoff++
* -ΔG ++
on
*
++

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

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

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