1 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
Oct 24, 2014
1
Lecture 8
Binding and kinetics
Antoine van Oijen
BCMP201 Spring 2008
Donald T. HayneBiological Thermodynamics
James Goodrich, Jennifer KugelBinding and Kinetics for Molecular Biologists
2
Goals
- Quantitative measurements of biological binding reactions
- Affinities- Cooperativity in binding- Kinetics
Practical use!!!
Assays: how much is bound?
• 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)
Protein-protein, protein-DNA, protein-ligand, …
3
Bimolecular interactions
A + B ABkon
koff
Binding is not all-or-nothing:
Portion of A and B will be bound, portion will be free
X Ykon
koff
!
d[Y]
dt= [X ] " k
on# [Y] " k
off= 0
Equilibrium
Reaction is in equilibrium when concentrations do not change:
(unimolecular reaction)
(mass action law)
4
Equilibrium
Reaction is in equilibrium when concentrations do not change:
!
d[AB]
dt= [A] " [B] " k
on# [AB] " k
off= 0 (mass action law)
A + B ABkon
koff
Equilibrium is reached when:
!
[A] " [B] " kon
= [AB] " koff
Equilibrium is still dynamic!!!
Binding (bimolecular reaction):
Equilibrium dissociation constant KD
Rearrange to define equilibrium dissociation constant KD:
!
KD
=k
off
kon
=[A] " [B]
[AB]
When [A]=Keq, 50% of B is bound to A
Equilibrium is reached when:
!
[A] " [B] " kon
= [AB] " koff
5
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})
!
KD
=k
off
kon
=[A] " [B]
[AB]
koff: {s-1}
kon: {M-1·s-1 }
Rate constants:
Where does this KD come from?
From Lecture 5:
6
How to measure KD ?
!
KD
=[A] " [B]
[AB]Measure [A], [B], and [AB]?
Introducing [A]Total=[A]+[AB]:
!
[AB]
[A]Total
=[B]
KD
+ [B]
D
Figure from: G
oodrich, Kugel
Experimental considerations
• [A] constant; titrate B• Measure fraction bound
!
[AB]
[A]Total
=[B]free
Keq + [B]free
If [A]Total << KD, then [B]≈[B]+[AB]
No need to measure [B],Just take [B]Total!
Figure from: G
oodrich, Kugel
7
Logarithmic versus linear display
As a corollary: Choose your titrations logarithmically!
1, 3, 10, 30, 100, 300 nM, or2, 4, 8, 16, 30, 60, 180, 360 nM, instead of50, 100, 150, 200, 250, 300 nM
Figure from: G
oodrich, Kugel
Example: Repressor binding to DNA
DNA + R DNA-Rkon
koff
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)
!
F =[R]
[R] + [R "DNA]=
[R][DNA
non]
[R "DNA]
[R][DNA
non]
[R "DNA]+ [R "DNA]
[DNAnon
]
[R "DNA]
=K
D
KD
+ [DNAnon
]=
10#4M
10#4M +10
-2M
= 0.01
Hardly any free repressor; almost all bound to nonspecific DNA!
KD≈10-10 M for operator DNA (specific binding)KD≈10-4 M for non-operator DNA (non-specific binding)
8
Protein
B BB
000 + BK
00B
Not Cooperative
00B + BK
0BB
Protein
B BB Cooperative
000 + BK
00B
00B + BτK
0BB
Non-cooperative versus cooperative
τ can be positive or negative (positive or negative cooperativity)
Cooperative binding
A + nB ABn
kon
koff
(perfect cooperativity)
Simplification:
!
KD
=[A] " [B]n
[ABn]
Rearrange (next Problem Set???):
!
logY
1"Y
#
$ %
&
' ( = n
H) log[B] " logK
D ,
where Y=[ABn]/[A]total
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Cooperative binding
!
logY
1"Y
#
$ %
&
' ( = n
H) log[B] " logK
D ,
where Y=[ABn]/[A]total
Figure from: G
oodrich, Kugel
Hemoglobin
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Reaction kinetics
Equilibrium thermodynamics does not provide anyinformation on rates of chemical changes!
Energy profile for ageneric chemical reaction:
Gibbs free energy (ΔG0) determines ratio of reactants/products(thermodynamic properties), activation energy (ΔG++) determinesrates (kinetics)
(dynamite versus nitroglycerin)
Figure from: H
aynie, Biological Therm
odynamics
++
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
Reaction rate:
!
J = "d[ATP]
dt= +
d[ADP]
dt= +
d[Pi]
dt
Figure from: H
aynie, Biological Therm
odynamics
11
Rate constant and order of reaction
!
J = k[A]n
Reaction rate/velocity is related to concentration of reactant:
n is order of reaction (often identical to stoichiometry)k is rate constant (don’t confuse with binding constant)
We saw that , so k will have:
Per second (s-1) as unit for 1st order reaction,Per molar per second (M-1s-1) as units for 2nd order reaction
!
J = "d[A]
dt
1st order reaction
1st order reaction A P
!
J = k[A]
!
J = "d[A]
dtCombining with gives:
!
"d[A]
dt= k[A]
!
1
[A]d[A] = "kdt
Integrate ( ):
!
ln[A] = ln[A]0 " kt
!
1
x" dx = ln x + C
!
[A]
[A]0
= e("kt)
Figure from: H
aynie, Biological Therm
odynamics
12
2nd order reaction
2nd order reaction 2A P
!
J = k[A]2
!
J = "d[A]
dtCombining with gives:
!
"d[A]
dt= k[A]2
!
1
[A]2d[A] = "kdt
Integrate ( ):
!
1
[A]=
1
[A]0
+ kt
!
1
x2
" dx = #1
x+ C
!
[A]
[A]0
=1
1+ kt
Figure from: H
aynie, Biological Therm
odynamics
1st and 2nd order reactions
1st order:
!
[A]
[A]0
=1
1+ kt
!
[A]
[A]0
= e("kt)
2nd order:
Figure from: H
aynie, Biological Therm
odynamics
13
Half-times and rate constants
Half time t1/2 is not the same as k-1 :
!
[A]
[A]0
= 0.50 = e("kt1/ 2 )
# " ln2 = "kt1/ 2 # t1/ 2 =ln2
k$
0.693
k
Temperature effects
Rates depend on temperature
Arrhenius:
!
k = Ae("#G
++ / RT)
A+B ABkon
koff
!
lnk = ln A "#G++
/ RT
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Reversible reaction
A + B ABkon
koff
!
d
dt[A "B] = k
on[A][B] # k
off[A "B]
Formation(2nd order)
Dissociation(1st order)
Under equilibrium, equals zero:
!
d
dt[A "B]
!
[A][B]
[A "B]=
koff
kon
= KD
In terms of free energies:
!
KD
=k
off
kon
=Ae
("#Goff
++ / RT)
Ae("#Gon
++ / RT)= e
("(#Goff
++"#Gon
++ ) / RT)= e
("#G0 / RT)
ΔG0= ΔGoff*-ΔGon
*
Figure from: H
aynie, Biological Therm
odynamics
!
[A][B]
[A "B]=
koff
kon
= KD
Relation between KD, kon/off, and ΔG
A+B
AB
++++ ++
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Rates of binding and dissociation
A+B ABkon
koff
Association rate for two objects with diffusion coefficients D1 and D2and 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 changeneeds to take place before binding
Example: Repressor binding to DNA
DNA + R DNA-Rkon
koff
!
d
dt[R "DNA] = k
on[R][DNA] # k
off[R "DNA]
Formation(2nd order)
Dissociation(1st order)
It takes 0.1 seconds to switch off gene expression in E.coli afterlactose depletion. What is kon?
!
d
dt[R "DNA] # k
on[R][DNA]
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???
16
1D sliding along DNA to speed up kon
BWH (Berg, Winter, von Hippel) model:Combine 3D diffusion (‘hopping’) with1D diffusion (‘sliding’). Scan short stretch of DNA by 1Dsearch, then jump to different area.
!
"L(# ) = D1D#
Length explored by one 1D sliding event:
(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 singlerepressor in half a minute
Searching 100 kb with only 1D sliding would takeTtotal = L2
total/D1D ≈ 3 hours!
Now we understand why 99% of repressor is bound to nonspecific DNA:They’re actively involved in the search process.
17
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)
18
Pulling at an RNA hairpin
Force-extension curve ofsingle RNA unfolding/folding
Liphardt et al., Science (2001)
Along the reaction coordinate, anamount of energy equal to forcetimes displacement is added
Force tilts free-energy diagrams
ΔG = -F·Δx
ΔG = ΔG0 - F·Δx
19
!
P(unfolded)
P(folded)= exp "
#G
kT
$
% &
'
( ) = exp "
#G0 "F#x
kT
$
% &
'
( )
Liphardt et al., Science (2001)
Pulling at an RNA hairpin
Liphardt et al., Science (2001)
Single-molecule kinetics:Direct observation of kopen and kclose
Pulling at an RNA hairpin: kinetics
20
Unfolding a riboswitch
Unfolding a riboswitch
21
Take-home message
Equilibrium constant K is related to free energy difference ΔG0
between initial and final state, rates k are related to free energydifferences ΔG‡ between initial/final state and transition state
!
[A][B]
[A "B]=
koff
kon
= KD
!
KD
=k
off
kon
=Ae
("#Goff
++ / RT)
Ae("#Gon
++ / RT)= e
("(#Goff
++"#Gon
++ ) / RT)= e
("#G0 / RT)
A+B
AB
++++ ++ΔG0= ΔGoff
*-ΔGon*