Molecular Interactions@ BIFI

ITC & SPRAdrián Velázquez Campoy

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