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):
kon koff

A+B

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

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

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

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

!

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

No need to measure [B], Just take [B]Total!

Figure from: Goodrich, Kugel

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

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

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

Non-cooperative versus cooperative

B
Not Cooperative

B Protein

000 + B 00B + B

K K

00B 0BB

Cooperative

B Protein

000 + B 00B + B

K K

00B 0BB

can be positive or negative (positive or negative cooperativity)

Cooperative binding Simplification: A + nB

kon koff

ABn
KD =

(perfect cooperativity)
[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 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 (G0) determines ratio of reactants/products (thermodynamic properties), activation energy (G++) 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:

J ="

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

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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 = " P gives:

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

" ! !

d[A] = k[A] dt !
1

1 d[A] = "kdt [A]

Figure from: Haynie, Biological Thermodynamics

Integrate ( " dx = ln x + C): x !

!

ln[A] = ln[A]0 " kt

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

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2nd order reaction

2nd order reaction 2A Combining J = " P

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

"
!

d[A] = k[A]2 dt !
1 1 dx = # + C): x x2

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

Figure from: Haynie, Biological Thermodynamics

Integrate ("

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

1st and 2nd order reactions

1st order:

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

2nd order:

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

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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
!

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Reversible reaction

A+B

kon koff

AB
d [A " B] = kon [A][B] # koff [A " B] dt
Formation (2nd order) Dissociation (1st order)

!
Under equilibrium,

d [A " B] dt

equals zero:

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

Relation between KD, kon/off, and G

Figure from: Haynie, Biological Thermodynamics

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

++

++ ++ G0= Goff* -Gon*

A+B

!
In terms of free energies:
++ ++ 0 koff Ae("#Goff / RT) KD = = = e("(#Goff "#Gon ) / RT) = e("#G / RT) ++ kon Ae("#Gon / RT) ++

AB

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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.

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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)

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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

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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)

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Unfolding a riboswitch

Unfolding a riboswitch

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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
++ ++ ++ G0= Goff* -Gon*

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

A+B AB

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

++

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