molecular interactions @ bifi itc & spr
DESCRIPTION
Molecular Interactions @ BIFI ITC & SPR. Adrián Velázquez Campoy. Molecular interaction techniques Dialysis Ultrafiltration Ultracentrifugation (sedimentation equilibrium and velocity) Chromatography (size exclusion, affinity) Membrane-filter binding Capillary electrophoresis - PowerPoint PPT PresentationTRANSCRIPT
Molecular interaction techniques
DialysisUltrafiltrationUltracentrifugation (sedimentation equilibrium and velocity)Chromatography (size exclusion, affinity)Membrane-filter bindingCapillary electrophoresisGel-shift electrophoresisUV/Visible spectroscopyFluorescence spectroscopy (correlation, intensity, polarization, anisotropy, FRET)Circular dichroismDynamic light scatteringNuclear magnetic resonance (HSQC, STD)IR spectroscopyRaman spectroscopyElectrochemistryIsothermal titration calorimetryDifferential scanning calorimetrySurface plasmon resonanceHydroxyl radical foot-printingProtease-digestion protectionMass spectrometryAtomic force microscopyX-ray diffractionX-ray absorption fine structureElectron microscopyBiological activity (e.g. enzymatic reaction)
Chemical cross-linkingTwo-hybrid systemsCo-precipitationWestern analysis
Fluorescence microscopy (correlation, FRET)Flow citometry (FRET)Confocal microscopy
Is there interaction between two biomolecules? Yes/No
What is the stoichiometry? n
How strong is the interaction? G, Ka
How fast does the interaction occurs? kon, koff
What intermolecular forces are involved? H, -TS, CP, +cond.
Is binding coupled to another binding process? nX, +molecules
Is binding coupled to a conformational change? H, -TS, CP
What functional groups are involved? +mutations
What is the interaction specificity? +mutations
Characterization of ligand binding
Dissection of binding energetics
Characterization of ligand specificity
Coupling between ligand events (homo- and heterotropic)
Allosteric control of protein function (homo- and heterotropic)
Ligand binding optimization
Drug development and design
Protein engineering and function redesign
Isothermal Titration Calorimetry
R S
T=TS-TR
dQ/dt
0 100 200 300
2.0
3.0
4.0
5.0
dQ/d
t (c
al/s
)
time (s)
f
o
t
tdt
dt
dQQ
TR Additional heat provided or subtracted during the thermal event in order to ensure T=0
0.0
0.5
1.0
1.5
2.0
2.5
3.00 30 60 90 120
dQ/d
t (c
al/s
)
time (min)
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
2.0
4.0
6.0
8.0
10.0
Q (
kcal
/mol
)
[Ligand]T/[Macromolecule]T
0.0
0.5
1.0
1.5
2.0
2.5
3.00 30 60 90 120
dQ/d
t (c
al/s
)
time (min)
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
2.0
4.0
6.0
8.0
10.0
Q (
kcal
/mol
)
[Ligand]T/[Macromolecule]T
H
n
Ka
MLLM
Experimental considerations:
ThermostatizationEquilibrium (absence of kinetic effects)Physiological/stabilizing/informative conditionsSolvent composition (co-solutes/co-solvents)Purity of reactants (chemical and conformational)Everything gives a heat signalDirect and reverse titrationsCalibration (electrical or chemical)
Concentrations: (rule of thumb...)
[M]0 = 5 – 20 M 2 ml[L]0 = (10 – 20) n [M]0 0.5 ml
0.0 0.5 1.0 1.5 2.0 2.5 3.0
-20
-16
-12
-8
-4
0
-8
-4
0
0 30 60 90 120 150 180 210
time (min)
dQ
/dt
(ca
l/s)
[2'CMP]T/[RNase A]
T
Q (
kca
l/mo
l of
inje
cta
nt)
Bovine Pancreatic Ribonuclease A2’CMP
Ka 2.9·106 M-1
H -19.3 kcal/moln 1.02
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
0.0
2.0
4.0
6.0
8.0
10.0
0.0
0.5
1.0
0 30 60 90 120 150 180 210
time (min)
dQ
/dt (
cal/s
)
[PPT]T/[STI]
T
Q (
kcal/m
ol o
f in
ject
ant)
Soybean Trypsin InhibitorPancreatic Porcine Trypsin
Ka 1.5·106 M-1
H 8.4 kcal/moln 1.2
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
2.0
4.0
6.0
8.0
10.0
Q (
kcal
/mol
of
inje
ctan
t)
[Ligand]T/[Macromolecule]
T
0.0 0.5 1.0 1.5 2.0 2.5 3.0
[Ligand]T/[Macromolecule]
T
0.0 0.5 1.0 1.5 2.0 2.5 3.0
[Ligand]T/[Macromolecule]
T
time (min)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
time (min)
dQ/d
t (c
al/s
)
time (min)
0.5[M]K
M10K
Ta
14a
50[M]K
M10K
Ta
16a
5000[M]K
M10K
Ta
18a
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0 30 60 90 120 150 180 210
time (min)
[3'CMP]T/[RNase A]
T
0.0 0.5 1.0 1.5 2.0 2.5 3.0
-20
-16
-12
-8
-4
0
-8
-4
0
0 30 60 90 120 150 180 210
time (min)dQ
/dt (c
al/s
)
[2'CMP]T/[RNase A]
T
Q (
kcal
/mol
of
inje
ctan
t)
ON
N
O
OHO
P
NH2
OH
OH
HOCH2
O
ON
N
O
NH2
HOCH2
OH O
P OH
OH
O
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0 30 60 90 120 150 180 210 240 270
time (min)
[5'CMP]T/[RNase A]
T
ON
N
O
NH2
OH OH
OP
OH
OH
O
Ka (M-1) H (kcal/mol)
2’CMP 2.9·106 M-1 -19.3
3’CMP 2.4·105 M-1 -18.6
5’CMP 4.2·103 M-1 -16.3
0 1 2 3 4 5 6
-20
-15
-10
-5
0
-2
-1
0
1
0 30 60 90 120 150 180 210
time (min)
dQ/d
t (
cal/s
)
[FMN]T/[FADS]
T
Q (
kcal
/mol
of
inje
ctan
t)
0 1 2 3 4 5 6
-20
-15
-10
-5
0
-2
-1
0
1
0 30 60 90 120 150 180 210
time (min)
dQ/d
t (
cal/s
)
[FMN]T/[FADS:ADP]
TQ
(kc
al/m
ol o
f in
ject
ant)
0 1 2 3 4 5
-20
-15
-10
-5
0
-2
-1
0
1
0 30 60 90 120 150
time (min)
dQ/d
t (
cal/s
)
[FMN]T/[FADS:ADP]
T
Q (
kcal
/mol
of
inje
ctan
t)
0 1 2 3 4
-20.0
-15.0
-10.0
-5.0
0.0
-2
-1
0
1
0 30 60 90 120 150 180 210
time (min)dQ
/dt
(ca
l/s)
[FMN]T/[FADS:ADP]
T
Q (
kcal
/mol
of
inje
ctan
t)
ADP 0 mMMgCl2 0 mM
ADP 0.5 mMMgCl2 0 mM
ADP 0 mMMgCl2 10 mM
ADP 0.5 mMMgCl2 10 mM
FAD Synthetase
Frago et al. (2009). Journal of Biological Chemistry 284 6610-6619
0 1 2 3 4 5
0
2
4
6
Q (
kca
l/mo
l of
inje
cta
nt)
Molar Ratio
1 5.5·104 M-1
H1 -1.9 kcal/mol2 4.2·109 M-2
H2 9.9 kcal/mol
42/12 5.4
nHill 1.40
cAMP Receptor Protein + cAMP
Gorshkova et al. (1995). Journal of Biological Chemistry 270 21679-21683
0.0 0.5 1.0 1.5 2.0
0.0
0.2
0.4
0.6
0.8
1.0
[ML
i] / [
M] T
nLB
ML
M ML2
20 25 30
-15
-12
-9
-6
-3
0
amprenavir TMC-126
1
H (
kcal
/mol
)
Temperature (°C)
-440 cal/Kmol
-350 cal/Kmol
Ohtaka et al. (2002). Protein Science 11 1908-1916
HIV-1 Protease
PP T
ΔHΔC
area surface accesible solvent in changeΔCP
0 2 4 6 8-14
-12
-10
-8
-6
amprenavir TMC-126
1
H (
kcal
/mol
)
Hion,B
(kcal/mol)
nH = 0.02H0 = 6.9 kcal/mol
nH = 0.39H0 = 12.0 kcal/mol
Ohtaka et al. (2002). Protein Science 11 1908-1916
HIV-1 ProteaseBion,H
0
Ha
ΔHnΔHΔH
npH
logK
H = -6.3 kcal/molpKa
F = 6.0 pKaC = 6.6
pKaF = 4.8 pKa
C = 2.9
Velazquez-Campoy et al. (2000). Protein Science 9 1801-1809
nH = -0.7H0 = -2.5 kcal/mol
nH = -0.09H0 = -4.7 kcal/mol
HIV-1 PR WT
HIV-1 PR V82F/I84V
KNI-529
KNI-272
KNI-272
HIV-1 Protease
Guzman-Casado et al. (2002). International Journal of Biological Macromolecules 31 45-54
Human Fibroblast Growth FactorHeparin
xa n
log[X]
logK
0.0
0.3
0.6
0.90 30 60 90 120 150 180 210
time (min)
dQ
/dt
(ca
l/s)
0 20 40 60 80 100 120
0.0
1.0
2.0
3.0
4.0
[-Chymotrypsin]T (M)
Q (
kca
l/mo
l of
inje
cta
nt)
Burrows et al. (1994). Biochemistry 33 12741-12745Velazquez-Campoy et al. (2004). Methods in Molecular Biology 261 35-54Belo et al. (2008). Proteins 70 1475-1487
Kd 53 MHd 5.5 kcal/mol
Bovine -Chymotrypsin
Cliff et al. (2005). Journal of Molecular Biology 346 717-732
Tetratricopeptide Repeat Domain (PP5) + MEEVD
TPR WT TPR G83N
TPR WT
TPR G83N
G
-TS
H
G
-TS
H
ITC: Advantages
• Complete thermodynamic characterization: H, Ka, n, G, and S
• Direct determination of the binding enthalpy (with no additional assumptions or models; no van’t Hoff enthalpy determination)
• Universal signal (heat), and high sensitivity (Q ~ cal)
• Absence of reporter labels (chromophores, fluorophores, etc.)
• Highly reproducible, and user-friendly with low maintenance cost
• Non-destructive technique (sample recovery)
• Interaction in solution (no need for immobilization)
• Experiments with unusual systems (e.g. dispersions, intact cells)
• Relatively fast and automatized technique (< 30 min/experiment)
ITC: Disadvantages
• Signal depends on H and concentrations. What if H is close to zero?
• What if affinity is very high or very low?
• Heat is a universal signal, so what are we observing in the cell?
• Need for additional experiments and control assays
• Relatively fast and user-friendly, but no high-throughput
• Very informative, but it consumes a big amount of sample
• Not often used for kinetics assays
• Slow binding processes may be overlooked
Surface Plasmon Resonance
C
OH
O C
O
O C
NH
OEDC/NHS
NO O
NH2
ligandligand
C
OH
O C
O
O C
NH
OEDC/NHS
NO O
SH
ligand
ligand
PDEA
C
NH
O
SS
SS
N
kon
MLLM
koffRUss
[L]K1
[L]KRURU
k
kK
k ,k
a
amaxss
off
ona
offon
SPR: Advantages
• No complete, but reasonable thermodynamic characterization: KA, n, G
• Universal signal (resonance units, RU), and high sensitivity
• Absence of reporter labels (chromophores, fluorophores, etc.)
• Need for very little sample
• Non-destructive technique (sample recovery)
• Exceptional for kinetic assays, also appropriate for equilibrium binding
• Experiments with unusual systems (e.g. dispersions, intact cells)
• Appropriate for high-throughput
SPR: Disadvantages
• Indirect determination of the binding enthalpy (with additional assumptions or models; van’t Hoff enthalpy determination)
• Signal depends on MW. What if analyte MW is very low?
• What if affinity is very high or very low?
• What if unspecific binding? Or improper immobilization?
• Very informative, but it requires numerous assays
• Often complaints regarding low reproducibility
• Often not user-friendly with high maintenance cost