microcalorimetry for the life sciences · microsoft powerpoint - damian.ppt author: frog created...

binding stability kinetics

Ultrasensitive Calorimetry for the Life SciencesTM

Microcalorimetry for the Life Sciences


kineticsstabilitybinding

Why Microcalorimetry?Microcalorimetry is universal detectorHeat is generated or absorbed in every chemical processIn-solutionNo molecular weight limitationsLabel-free Non-optical



Calorimetry in the Life SciencesBinding Studies

Quick and accurate affinitiesMechanism of action (MOA) screening and conformational changes Structure-function relationshipsSpecific vs. non-specific binding

KineticsKM, Vmax, kcatEnzyme Inhibition

Stability StudiesIntramolecular – e.g. protein unfoldingIntermolecular – e.g. solution optimization, CMCs



Isothermal Titration Calorimetry

VP-ITC

AutoITC

-8.33 0.00 8.33 16.67 25.00 33.33 41.67 50.00 58.33 66.67 75.002

4

6

8

10

12

14

16

Time (min)

µcal

/sec



Differential Scanning Calorimetry

VP-DSC

VP-Capillary DSC Platform



How Do They Work?Measuring Temperature Changes in Calorimetry



Isothermal Titration Calorimetry: A Method for Characterizing

Binding Interactions

Mixture of two components at a set temperatureHeat of interaction is measuredParameters measured from a single ITC experiment

AffinitiesBinding mechanismNumber of binding sitesKinetics



Isothermal Titration Calorimetry

-14

-12

-10

-8

-6

-4

-2

0

kcal

/mol

e of

inje

ctan

t

0 2 4Molar Ratio

Affinity

-8.33 0.00 8.33 16.67 25.00 33.33 41.67 50.00 58.33 66.67 75.002

4

6

8

10

12

14

16

Time (min)

µcal

/sec

Typical ITC Data

Stoichiometry

ΔHMechanism



ITC – Protein-Small Molecule Interaction

4-carboxybenzene-sulfonomide titrated into carbonic anhydrase IIN = 0.97KD = 730 nMΔH = -11.9 kcal/mol

Day, et al, Protein Sci. 11, 1017-1025 (2002)



ITC – Protein-Protein Interaction

Pielak and Wang, Biochemistry 40, 422-428 (2001)

A: Wild-type cytochrome c titrated into wild-type cytochrome c peroxidase

B: Mutant cytochrome c titrated into mutant cytochrome c peroxidase



Multiple Binding Sites

ITC shows differential binding of Mn(II) ions to WT T5 5’ nuclease

-2

0

2

4-10 0 10 20 30 40 50 60 70 80 90 100Time (min)

µcal

/sec

-0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0

0

2

Molar Ratio

kcal

/mol

e

n = 0.85Ka = 3.0 x 105 M-1

ΔH = -0.59 kcal mol-1

n = 1.3Ka = 1.0 x 104 M-1

ΔH = +1.6 kcal mol-1

Feng, et al, Nat. Struct. Mol. Biol. 11, 450-456 (2004)



Binding EnergeticsMechanism of Action (MoA)

Conformational changesH-bondingHydrophobic interactionsCharge-charge interactions

Multiprobe structure-activity relationship (SAR)Validate in-silico modellingSelectivity and adaptability of drugs



Binding Mechanism Same affinity but different binding mechanisms and specificity

-1 4

-1 2

-1 0

-8

-6

-4

-2

0

kcal

/mol

e of

inje

ctan

t

0 1 2 3 4



Enthalpic and Entropic Contributions to Binding Affinity

Enthalpy and Entropy make up the affinity (ΔG=-RTlnK)

-8

-6

-4

-2

0

2

4

6

kcal

mol

-1

ΔH

ΔG

TΔS

ΔG=ΔH-TΔS



Enthalpy and Entropy

EntropyHydrophobic interactionsWater releaseIon release Conformational changes

EnthalpyHydrogen bonding Protonation eventsMore specific



Energetic Signatures

A is enthalpy driven. Good H-bonding coupled to a conformational change

B is entropicallydriven. Hydrophobic Interactions and possibly ‘rigid body’

C is mildly enthalpic and entropic -100

-80

-60

-40

-20

0

20

40

60

ΔHΔG

-TΔS

kJ mol-1

A B C



Drug Discovery – Binding of Inhibitors to HIV-1 Protease

KNI-764KNI-272LopinavirAmprenavirRitonavirSaquinavirNelfinavirIndinavir

-20

-15

-10

-5

0

5

kcal

/mol

e

ΔG

ΔH

−TΔS

Ohtaka, et al. Protein Sci. 11, 1908-1916 (2002)



Enzyme kinetics and ITC

ITC measures thermal power (dQ/dt)



Enzyme kineticsRate =

Vo·

dQ/dt



ITC and Binding - Summary

Quick and Easy AffinitiesMechanism of ActionSAR-Structure Activity RelationshipsDrug designMulti-probe technique detects contributions that ‘affinity only’ methods may missEnzyme kinetics



Differential Scanning CalorimetryTypical Data



Minimum Protein for DSC - Lysozyme

Kholodenko and Freire, Anal. Biochem. 270, 336-338 (1999)

25 μg/ml



Data Interpretation

30 40 50 60 70 80 90

0

2

4

6

8

10

12

14

Cp

(kca

l/mol

e/o C)

Temperature ( oC)

TM Shelf-Life/ Stability

Oligomers

Binding

ΔCp}

ΔH Stabilizing Forces/ Energetics



Data Interpretation

30 40 50 60 70 80 90

0

2

4

6

8

10

12

14

Cp

(kca

l/mol

e/o C)

Temperature ( oC)

TM

ΔCp}

ΔH

Shelf-Life/ Stability

Oligomers

Binding

Stabilizing Forces/ Energetics



Stability Intrinsic molecular stability

Thermodynamic characterization of macromolecular unfolding- proteins, nucleic acids, lipids, Domain structure identificationAssessment of viability and/or ‘crystallization potential’ of protein constructs and mutants

Extrinsic molecular stabilityFormulation studies – effect of different excipients, preservatives BindingMembrane and lipid studies



DSC –Protein Stability and Mutant Characterization

Sot, et al, J. Biol. Chem. 278, 32083 - 32090 (2003)



Domain Identification

Structural organization of biomolecules

Protein has a least two structural domains

O’Brien, et al, Biophys J. 70, 2403-2407 (1996)



Formulations/Protein Storage

CD40 ligand has a longer shelf-life at pH 6-7.DSC experiment could be completed in 1 day as opposed to 8 days by size exclusion chromatography

Remmele and Gombotz, BioPharm 13, 36-46 (2000)



Binding

20 40 60 80 100 120-0.00135

-0.00130

-0.00125

-0.00120

-0.00115

-0.00110

-0.00105

-0.00100

Cp(

cal/o C

)

Temperature (oC)

FBS at 60 μM Plus VAF955 and 1827 (at 1:1 and 1:20 [Ratios])

No ligand

1827(1:1)

1827 (1:20)

VAF955 (1:20)

VAF (1:1)

HTS validation OR Ligand Identification



Antibody StabilityEffects of Glycosylation

Native Partially Deglycosylated Deglycosylated

Mimura, et al, J. Biol. Chem. 276, 45539-45547 ( 2001)



Lipid/Membrane systems

Uptake of proteins into lipid membrane causes peak broadening

Lipid phase transitions

Broad Narrow



Microcalorimetry Summary

Affinities and Binding EnergeticsMechanisms of BindingStoichiometryDetermination of Active ConcentrationEnzyme KineticsThermodynamic StabilityFormulations and Drug delivery



Conclusion

Microcalorimetry is one of the most versatile technologies available for

characterization and analysis of biological molecules

microcalorimetry for the life sciences · microsoft powerpoint - damian.ppt author: frog created...

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