microcalorimetry for the life sciences · microsoft powerpoint - damian.ppt author: frog created...
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![Page 1: Microcalorimetry for the Life Sciences · Microsoft PowerPoint - Damian.ppt Author: Frog Created Date: 9/23/2006 5:59:50 PM](https://reader033.vdocument.in/reader033/viewer/2022051608/603eda8fd8d0302ad46fdf0c/html5/thumbnails/1.jpg)
binding stability kinetics
Ultrasensitive Calorimetry for the Life SciencesTM
Microcalorimetry for the Life Sciences
![Page 2: Microcalorimetry for the Life Sciences · Microsoft PowerPoint - Damian.ppt Author: Frog Created Date: 9/23/2006 5:59:50 PM](https://reader033.vdocument.in/reader033/viewer/2022051608/603eda8fd8d0302ad46fdf0c/html5/thumbnails/2.jpg)
Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
Why Microcalorimetry?Microcalorimetry is universal detectorHeat is generated or absorbed in every chemical processIn-solutionNo molecular weight limitationsLabel-free Non-optical
![Page 3: Microcalorimetry for the Life Sciences · Microsoft PowerPoint - Damian.ppt Author: Frog Created Date: 9/23/2006 5:59:50 PM](https://reader033.vdocument.in/reader033/viewer/2022051608/603eda8fd8d0302ad46fdf0c/html5/thumbnails/3.jpg)
Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
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
![Page 4: Microcalorimetry for the Life Sciences · Microsoft PowerPoint - Damian.ppt Author: Frog Created Date: 9/23/2006 5:59:50 PM](https://reader033.vdocument.in/reader033/viewer/2022051608/603eda8fd8d0302ad46fdf0c/html5/thumbnails/4.jpg)
Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
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
![Page 5: Microcalorimetry for the Life Sciences · Microsoft PowerPoint - Damian.ppt Author: Frog Created Date: 9/23/2006 5:59:50 PM](https://reader033.vdocument.in/reader033/viewer/2022051608/603eda8fd8d0302ad46fdf0c/html5/thumbnails/5.jpg)
Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
Differential Scanning Calorimetry
VP-DSC
VP-Capillary DSC Platform
![Page 6: Microcalorimetry for the Life Sciences · Microsoft PowerPoint - Damian.ppt Author: Frog Created Date: 9/23/2006 5:59:50 PM](https://reader033.vdocument.in/reader033/viewer/2022051608/603eda8fd8d0302ad46fdf0c/html5/thumbnails/6.jpg)
Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
How Do They Work?Measuring Temperature Changes in Calorimetry
![Page 7: Microcalorimetry for the Life Sciences · Microsoft PowerPoint - Damian.ppt Author: Frog Created Date: 9/23/2006 5:59:50 PM](https://reader033.vdocument.in/reader033/viewer/2022051608/603eda8fd8d0302ad46fdf0c/html5/thumbnails/7.jpg)
Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
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
![Page 8: Microcalorimetry for the Life Sciences · Microsoft PowerPoint - Damian.ppt Author: Frog Created Date: 9/23/2006 5:59:50 PM](https://reader033.vdocument.in/reader033/viewer/2022051608/603eda8fd8d0302ad46fdf0c/html5/thumbnails/8.jpg)
Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
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
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Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
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)
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Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
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
![Page 11: Microcalorimetry for the Life Sciences · Microsoft PowerPoint - Damian.ppt Author: Frog Created Date: 9/23/2006 5:59:50 PM](https://reader033.vdocument.in/reader033/viewer/2022051608/603eda8fd8d0302ad46fdf0c/html5/thumbnails/11.jpg)
Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
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)
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Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
Binding EnergeticsMechanism of Action (MoA)
Conformational changesH-bondingHydrophobic interactionsCharge-charge interactions
Multiprobe structure-activity relationship (SAR)Validate in-silico modellingSelectivity and adaptability of drugs
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Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
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
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Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
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
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Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
Enthalpy and Entropy
EntropyHydrophobic interactionsWater releaseIon release Conformational changes
EnthalpyHydrogen bonding Protonation eventsMore specific
![Page 16: Microcalorimetry for the Life Sciences · Microsoft PowerPoint - Damian.ppt Author: Frog Created Date: 9/23/2006 5:59:50 PM](https://reader033.vdocument.in/reader033/viewer/2022051608/603eda8fd8d0302ad46fdf0c/html5/thumbnails/16.jpg)
Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
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
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Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
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)
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Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
Enzyme kinetics and ITC
ITC measures thermal power (dQ/dt)
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Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
Enzyme kineticsRate =
Vo·
dQ/dt
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Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
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
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Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
Differential Scanning CalorimetryTypical Data
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Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
Minimum Protein for DSC - Lysozyme
Kholodenko and Freire, Anal. Biochem. 270, 336-338 (1999)
25 μg/ml
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Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
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
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Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
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
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Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
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
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Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
DSC –Protein Stability and Mutant Characterization
Sot, et al, J. Biol. Chem. 278, 32083 - 32090 (2003)
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Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
Domain Identification
Structural organization of biomolecules
Protein has a least two structural domains
O’Brien, et al, Biophys J. 70, 2403-2407 (1996)
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Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
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)
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Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
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
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Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
Antibody StabilityEffects of Glycosylation
Native Partially Deglycosylated Deglycosylated
Mimura, et al, J. Biol. Chem. 276, 45539-45547 ( 2001)
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Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
Lipid/Membrane systems
Uptake of proteins into lipid membrane causes peak broadening
Lipid phase transitions
Broad Narrow
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Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
Microcalorimetry Summary
Affinities and Binding EnergeticsMechanisms of BindingStoichiometryDetermination of Active ConcentrationEnzyme KineticsThermodynamic StabilityFormulations and Drug delivery
![Page 33: Microcalorimetry for the Life Sciences · Microsoft PowerPoint - Damian.ppt Author: Frog Created Date: 9/23/2006 5:59:50 PM](https://reader033.vdocument.in/reader033/viewer/2022051608/603eda8fd8d0302ad46fdf0c/html5/thumbnails/33.jpg)
Ultrasensitive Calorimetry for the Life SciencesTM
kineticsstabilitybinding
Conclusion
Microcalorimetry is one of the most versatile technologies available for
characterization and analysis of biological molecules