binding of ligands to a macromolecule - heidelberg...
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Binding of ligands to a macromolecule
• General description of ligand binding–the esssentials–thermodynamics–Adair equation
• Simple equilibrium binding–stoichiometric titration–equilibrium binding/dissociation constant
• Complex equilibrium binding–cooperativity–Scatchard plot and Hill Plot–MWC and KNF model for cooperative binding
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∆G of an reaction in equilibrium
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The mass equation law for binding of a protein P to its DNA D
binding of the first proteins with the dissociation constant K1
Dfree, concentration free DNA; Pfree, concentration free protein
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What is the meaning of the dissociation constant forbinding of a single ligand to its site?
2. KD gives the concentration of ligand that saturates 50% of the sites
(when the total sit concentration ismuch lower than KD)
3. Almost all binding sites are saturated if the ligand
concentration is 10 x KD
1. KD is a concentration and has units of mol per liter
4. The dissociatin constant KD is related to Gibbs free energy ∆G
by the relation ∆G = - R T ln(KD)
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Increasing complexity of binding
all binding sites areequivalent and independent
cooperativity heterogeneity
all binding sites areequivalent and not independent
cooperativityheterogeneity
all binding sites arenot equivalent and not independent
all binding sites areindependent but not equivalent
simple
difficult
verydifficult
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Titration of a macromolecule D with n binding sitesfor the ligand P which is added to the solution
free ligand Pfree (M)
degr
ee o
f bin
ding
ν
n n binding sitesoccupied
∆Xmax
∆X
0
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Schematic view of gel electrophoresisto analyze protein-DNA complexes
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“Gel shift”: electorphoretic mobility shift assay (“EMSA”) for DNA-binding proteins
Free DNA probe*
*Protein-DNA complex
1. Prepare labeled DNA probe2. Bind protein3. Native gel electrophoresis
Advantage: sensitive, fmol DNA
Disadvantage: requires stable complex; little “structural” information about which protein is binding
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EMSA of Lac repressor binding to operator DNAFrom (a) to (j) the concentrationof lac repressor is increased.
Complexes with
Free DNA
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Measuring binding constants for lambda repressor on a gel
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Principle of filter-binding assay
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A macromolecule is dialyzed against a solution of ligand. Upon reaching equilibrium, the ligand concentration is measured inside and outside the dialysis chamber. The excess ligand inside the chamber corresponds to bound ligand.
- direct measurement of binding
-non-specific binding will obscure results, work at moderate ionic strength (≥50 to avoid the Donnan Effect (electrostatic interactions between the macromolecule and a charged ligand.
- needs relatively large amounts of material
Binding measurments by equilibrium dialysis
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Analysis of binding of RNAP·σ54 to a promoter DNA sequenceby measurements of fluorescence anisotropy
Rho
Rho
+RNAP·σ54
promoter DNA
RNAP·σ54-DNA-Komplex
Kd
free DNA with a fluorophorewith high rotational diffusion
-> low fluorescence anisotropy rmin
RNAP-DNA complexwith low rotational diffusion
-> high fluorescence anisotropy rmax
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How to measure binding of a protein to DNA?One possibility is to use fluorescence anisotropy
z
y
x sample
I⊥
verticalexcitation
filter/mono-chromator
polarisatorIII
filter/monochromator
polarisator
measured fluorescenceemission intensity
Definition of fluorescenceanisotropy r
The anisotropy r reflects the rotational diffusion of a fluorescent species
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Measurements of fluorescence anisotropy tomonitor binding of RNAP·σ54 to different promoters
Vogel, S., Schulz A. & Rippe, K.
0
0.2
0.4
0.6
0.8
1
0.01 0.1 11 0 100 1000
nifH nifLglnAp2
RNAP σ54 (nM)
200 mM K-Acetate
Ptot = KD
θ = 0.5
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Titration of a macromolecule D with n binding sitesfor the ligand P which is added to the solution
free ligand Pfree (M)
degr
ee o
f bin
ding
ν
n n binding sitesoccupied
∆Xmax
∆X
0
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Example: binding of a protein P to a DNA-fragment D with one or two binding sites
binding of the first proteins withthe dissociation constant K1
Dfree, concentration free DNA; Pfree, concentration free protein;DP, complex with one protein; DP2, complex with two proteins;
binding of the second proteins withthe dissociation constant K2
alternative expression
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Definition of the degree of binding ν
degree of binding ν ν for one binding site ν for two binding sites
ν for n binding sites (Adair equation)!
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Binding to a single binding site: Deriving an expressionfor the degree of binding ν or the fraction saturation θ
from the Adair equation we obtain:
Often the concentration Pfree can not be determined but the total concentration of added protein Ptot is known.
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Stoichiometric titration to determinethe number of binding sites
To a solution of DNA strands with a single binding site small amounts of protein P are added. Since the binding affinity of the protein is high (low KD value as compared to the total DNA concentration) practically every protein binds as long as there are free binding sites on the DNA. This is termed “stoichiometric binding” or a “stoichiometric titration”.
ν or
θ
0
0.2
0.4
0.6
0.8
1
0 1·10 -10
Ptot (M)
equivalence point1 protein per DNA
2·10 -10
Dtot = 10-10 (M)
KD = 10-14 (M)
KD = 10-13 (M)
KD = 10-12 (M)
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Binding to a single binding site. Titration of DNA with aprotein for the determination of the dissociation constant KD
Ptot or Pfree (M)
ν or
θ
0
0.2
0.4
0.6
0.8
1
0 2 10-9 4 10-9 6 10-9 8 10-9 1 10-8
Ptot = KD
ν or θ = 0.5
Dtot = 10-10 (M)
KD = 10-9 (M)
KD = 10-9 (M)
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Lac repressor binding to DNA-
linking structure and thermodynamics
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Organization of the genes regulated by Lac repressor, a transcription repressor protein in the bacterium E. coli
Lac repressor binds to the
operators O1, O2 and O3
O3
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Molecular structure of E. coli lac repressor dimer
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Molecular structure of E. coli lac repressor tetramer
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Lac repressor binding sites
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Lac repressor head piece (1-62) bound to SynL sequence
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Lac repressor head piece (1-62) bound to the natural operator O1
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Binding titrations of symmetrical operator site with Lac repressor measured by filter binding assay
0.15 M K+, 297 K
0.30 M K+, 297 K
0.15 M K+310 K
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∆SPE: Favorable displacement of ions from the DNA
– – – – – – – – – – – – – – – – – – – – – – – – – – –
�
ΔSPE = 20 to 60 cal K-1 mol-1
+ + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + +– – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – – – – – – – – – – – – – – – – – – – – – + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + + + + + + + + + +– – – – – – – – – – – – – – – – – – – – – – – – – – –
+
+
+++
+
+
++ +
+
+
++ + + + + + + + + +
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The influence of binding one ligand (ion) on binding of another ligand (protein) to the DNA is referred to as binding “linkage”
specific binding of TBP to different promoter sequences
number of salt bridges between DNA phosphates and positive amino acids
fraction of chargeneutralized = 0.88
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Including the binding of ions in the reaction
�
P + D→←PD + mM+
Upon binding to the DNA D the protein P interacts with n phosphates so that m = n·ψ cations M+ are displaced:
�
K =PD ⋅ M+[ ]m
P ⋅D
However, we derive K from the concentrations of P, D and PD, thus
�
Kobs =PD[ ]
P[ ] ⋅ D[ ] = K
M+[ ]m
�
log Kobs = log K −m log M+[ ]
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Lac repressor binding involves 6-7 (specific) and 11 (nonspecific) charge-charge interactions
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The temperature dependence of the binding constants reveals ∆H and ∆S in a van’t Hoff plot if ∆H and ∆S are independent of temperature
From the slope of ln Keq vs. 1/T (usually from 0 to 40 °C) one can determine the ∆H and from extrapolaDon also ∆S . Is the van’t Hoff plot curved then ∆H is temperature dependent and it can be determined from the derivaDve.
1-oK1
eqKln
SoΔ
- HSlope=
T!
" (lnKeq )" (1/T )
=#$HR
!
lnKeq="#HRT
+#SR
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The heat capacity CP describes the temperature dependence of ∆H and ∆S
.
Enth
alpi
e H
Temperatur T
Steigung ist Wärmekapazität Cp�
CP =∂H∂T=T∂S∂T
�
H(T2)−H(T1)=CP ⋅(T2−T1)
or if CP is constant
which is a good approximation for the narrow interval from 0 to 40 °C usually studied
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Relation between ∆CP, ∆G and Keq for binding
�
ΔH (T ) =ΔCP ⋅(T −TH )
ΔS(T ) =ΔCP ⋅lnTTS
⎛
⎝ ⎜
⎞
⎠ ⎟
ΔG(T ) =ΔCP ⋅(T −TH ) − T ⋅ΔCP ⋅lnTTS
⎛ ⎝ ⎜
⎞ ⎠ ⎟
⇔
lnKeq = ΔCP
R⋅ THT
− 1 − ln TST
⎛ ⎝ ⎜
⎞ ⎠ ⎟
⎡
⎣ ⎢
⎤
⎦ ⎥
�
ΔH(TH)= 0 and ΔS(TS)= 0 ⇒
For two characteristic temperature TH and TS with
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ΔCp vs ΔAnp for protein folding
There is a linear correlation between the heat capacity change for protein unfolding and the buried non-polar surface area.This relationship is identical to that seen for the transfer of hydrocarbons from aqueous solution to the pure liquid phase
From Livingstone JR, Spolar RS, Record MT Jr. Biochemistry. 1991 Apr 30;30(17):4237-44
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Temperature dependence of equilibirium binding constant for specific binding of lac repressor to the operator DNA
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Temperature dependence of Kd for specific/nonspecific binding of lac repressor => less induced folding in the unspecific complex
specific binding vs.unspecific binding
NOD(per duplex)
NOD(per site)
operatormutations
operatorsequenceOsym
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Temperature dependence of Kd for specificbinding of LacI repressor => induced folding
specific binding to operator
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The hinge region (50-62 in red) of Lac-DBD is foldedonly in the specific complex with DNA
straight DNA curved DNA
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Specific (left) and nonspecific (right) protein-DNA contacts of Lac-DBD repressor with DNA
red:hydrogen bonds
green:hydrophobiccontacts dashed blue:electrostatic contacts
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Schematic models of the specific (RO) and nonspecific (RD) complexes of Lac repressor
- Small arrows denote specific hydrogen bonding in the protein binding site. That are established in the specific complex upon folding of the hinge region- Plus signs (+) denote basic side chains located in and around the same site. In the “down” position these groups are in “interactive contact” with the underlying dsDNA, and in the “up” position these contacts are broken.- RO complex: 7 hydrogen bonds with the base pairs of the DNA operator site, only 6 electrostatic interactions with the charged DNA backbones.- RD complex: 11 charge-charge interactions with the dsDNA backbone, but all the specific interactions with the DNA base pairs have been “withdrawn.”- curvature of DNA in the specific complex
RO and RD conformations are dynamic and interconvert withrate constants kRO and kRDtransient curvature of DNA alsoin unspecific complex?