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Isothermal Titration Calorimetry: Principles and Experimental Design

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Isothermal Titration Calorimetry: Principles and Experimental Design Isothermal titration calorimetry: Principles and experimental design Agenda Overview of Isothermal Titration Calorimetry ITC experimental design Data analysis Troubleshooting 2 GE Title or job number 11/2/2012 What is isothermal titration calorimetry (ITC) A direct measurement of the heat generated or absorbed when molecules interact Microcalorimetry offers enhanced information content Label-free In-solution No molecular weight limitations Optical clarity unimportant Minimal or no assay development 4 GE Title or job number 11/2/2012 How Do ITCs Work? The DP is a measured power differential between the reference and sample cells necessary to S R maintain their temperate difference at close to zero DT DP Reference Calibration Heater Sample Calibration Heater DT~0 Cell Main Heater 5 GE Title or job number 11/2/2012 Performing an ITC experiment Ligand in syringe Macromolecule in sample cell Syringe Heat of interaction is measured Parameters measured from a single ITC experiment: Affinity - KD Energy (Enthalpy) - DH Reference Cell Sample Cell Number of binding sites - n 6 GE Title or job number 11/2/2012 ITC – Before titration Ligand – in syringe Macromolecule in ITC cell 7 GE Title or job number 11/2/2012 Titration begins: First injection Ligand in syringe Macromolecule in cell Macromolecule-ligand complex 5 As the first injection is made, all injected ligand is bound to target macromolecule. 0 8 GE Title or job number 11/2/2012 Return to baseline 5 The signal returns to baseline before the next injection. 0 9 GE Title or job number 11/2/2012 Second injection 5 As a second injection is made, again all injected ligand becomes bound to the target. 0 10 GE Title or job number 11/2/2012 Second return to baseline 5 Signal again returns to baseline before next injection. 0 11 GE Title or job number 11/2/2012 Injections continue As the injections 5 continue, the target becomes saturated with ligand, so less binding occurs and the heat change starts to decrease. 0 12 GE Title or job number 11/2/2012 Injections continue As the injections 5 continue, the target becomes saturated with ligand so less binding occurs and the heat change starts to decrease. 0 13 GE Title or job number 11/2/2012 End of titration When the macromolecule is 5 saturated with ligand, no more binding occurs, and only heat of dilution is 0 observed. 14 GE Title or job number 11/2/2012 Experimental results 5 = 1/KD 0 15 GE Title or job number 11/2/2012 MicroCal™ ITC systems MicroCal™ VP-ITC MicroCal™ iTC200 MicroCal™ Auto-iTC200 • 1400 L cell • Sensitive • Unattended operation • Manual sample • Fast • Up to 75 samples/day (using single injection loading • Easy to use method) • Up to 5 • KD from mM to nM • K from mM to nM samples/day D • 200 µL cell • Sample cell is 200 µL • Upgradable to full • Easy to use automation • 96-well plate format 16 GE Title or job number 11/2/2012 Why ITC? Heat is a fundamental natural property… A single titration can yield information on: Overall binding affinity Hydrogen bonds and van der Waals interactions Hydrophobic and conformational effects Stoichiometry calorimetry is a direct readout 18 GE Title or job number 11/2/2012 Stoichiometry Number of ligand binding sites per macromolecule If one binding site the stoichiometry is 1 By convention a “Ligand” has one binding site A “Macromolecule” can have more than one binding site 19 GE Title or job number 11/2/2012 Effective Binding Affinity Range KD in mM to nM range Weak binding – low C-value method Tight binding - minimize injection volume and concentration or use competitive (displacement) binding procedure and fitting model 20 GE Title or job number 11/2/2012 Thermodynamics KB – binding constant [L] x [M] K = 1/ K = D B [ML] DG = RT lnKD DG = DH - T DS ` 21 GE Title or job number 11/2/2012 Free energy change DG is change in free energy DG 0 for spontaneous process More negative DG, higher affinity 22 GE Title or job number 11/2/2012 Enthalpy change DH – measure of the energy content of the bonds broken and created. The dominant contribution is from hydrogen bonds. Negative value indicates enthalpy change favoring the binding Solvents play a role 23 GE Title or job number 11/2/2012 Entropy change DS – positive for entropically driven reactions Hydrophobic interactions Solvation entropy (favorable) due to release of water Conformational degrees of freedom (unfavorable) 24 GE Title or job number 11/2/2012 Microcalorimetry provides a total picture of binding energetics Overall binding affinity KD correlates with IC50 or EC50. This is directly related to ∆G, the total free binding energy ∆H, enthalpy is indication of changes in hydrogen and van der Waals bonding -T∆S -T∆S, entropy is indication of changes ∆H in hydrophobic interaction and conformational changes N, stoichiometry indicates the ratio of ligand-to-macromolecule binding DG = DH -TDS 25 GE Title or job number 11/2/2012 Same affinity, different energetics! All three interactions have the same binding energy (∆G) Unfavorable 10 A. Good hydrogen bonding with 5 unfavorable conformational 0 change ∆G -5 ∆H kcal/mole -T∆S B. Binding dominated by -10 hydrophobic interaction -15 -20 C. Favorable hydrogen bonds A B C and hydrophobic interaction Favorable ITC results are used to get insights into mechanism of binding 26 GE Title or job number 11/2/2012 How to get good ITC data C Values [M] 0 C = K -2 D -4 Example: -6 0.05 0.5 -8 Kd = 100nM kcal/mole of injectant of kcal/mole 5 -10 50 -12 [M] = 100nM, C=1 -14 500 [M] = 5uM, C=50 -16 0.0 0.5 1.0 1.5 2.0 Molar Ratio [M]:[L] – 1:10 for n=1 28 GE Title or job number 11/2/2012 C Values tim e (m in) 0 30 60 90 120 150 0 -1 (a) µcal/s -2 0 -4 c = 5 -8 Kd = 1.2 µM (b) -12 c = 40 -16 kcal/mol of injected Ras 0.0 0.5 1.0 1.5 2.0 2.5 3.0 molar ratio (Ras / RalGDS-RBD) 29 GE Title or job number 11/2/2012 C Values in ITC C = {[M]tot / KD} * N C = 20-100 very good C = 10-500 good C = 1-5 and 500-1000 OK C = < 1 and > 1000 not wanted 30 GE Title or job number 11/2/2012 ITC experimental design High C Low C 0 00 0 -2 -2-2 10< [Protein]/K <100 -0.2 [Protein]/K >> 1000 D of injectant D 1 Fitted: N, KD, DH - Fitted: N, DH [Protein]/KD < 1 kcal/molekcal/mole of of injectant injectant kcal/mole of injectant N fixed -4-4 -0.4 -4 Fitted: KD, DH kcal kcal mol 0.0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 0 4 8 12 16 Molar Ratio Molar ratio 31 GE Title or job number 11/2/2012 ITC experimental design KD (Biacore) µM [Protein] µM [Compound] µM [Protein] / KD <0.5 10 100 >20 0.5-2 30 300 15-60 2-10 50 500 5-25 10-100 30 40*K 0.3-3 D Fixed stoichiometry >100 30 20*KD <0.3 34 GE Title or job number 11/2/2012 Low C Experiments Extending the applications of ITC – E.g. Fragment Based Drug Discovery Saving Protein 35 GE Title or job number 11/2/2012 C Values-Low 0 -1.1 -2 -1.2 -4 -1.3 -6 -1.4 -8 kcal/mole of injectant of kcal/mole kcal/mole of injectant of kcal/mole -1.5 -10 -12 -1.6 -14 -1.7 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 0 2 Molar Ratio Molar Ratio Note different scales 36 GE Title or job number 11/2/2012 C Values-Low 0 Increase ligand concentration but not the ‘valuable’ protein kcal/mole of injectant of kcal/mole -2 0 2 4 6 8 10 12 14 16 18 20 22 24 26 Molar Ratio 37 GE Title or job number 0--------------->26 11/2/2012 Competition Experiments Extend the affinity range that ITC can be used • Submillimolar (10-2) to picomolar (10-12) 38 GE Title or job number 11/2/2012 Competition Experiments 0 High C Experiment -2 Poor affinity estimates -4 -6 100% Binding 0% Binding -8 kcal/mole of injectant of kcal/mole -10 -12 -14 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 Molar Ratio 39 GE Title or job number 11/2/2012 Competitive (displacement) ITC Used to extend range of KB determined by ITC Tight binding ligand A and weak binding ligand B bind to same site on macromolecule 1st experiment: Ligand B titrated into macromolecule. Determine KB and DH 2nd experiment: Macromolecule + ligand B in cell, titrated with Ligand A. Ligand A displaces ligand B. Use displacement model for data analysis 40 GE Title or job number 11/2/2012 Tight Binders Protein Tight Ligand Competitor 0 0 -2 -2 -4 -4 Software -6 -6 -8 -8 kcal/mole of injectant of kcal/mole -10 kcal/mole of injectant of kcal/mole can ‘pull out’ the K -10 D -12 -12 -14 of the tight one -14 -16 -16 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 Molar Ratio Molar Ratio 41 GE Title or job number 11/2/2012 Weak Binders 0 -2 -4 + -6 -8 kcal/mole of injectant of kcal/mole -10 -12 -14 0 -16 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 Molar Ratio kcal/mole of injectant of kcal/mole -10 0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 Molar Ratio alone kcal/mole of injectant of kcal/mole -10 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 Molar Ratio Software can pull the Weak KD out 42 GE Title or job number 11/2/2012 Competition Experimental Design C = [cell]/KD,app KD,app= (1/KD,S)/(1+1/KD,W[W]) Where KD,S and KD,W are the affinity of the strong and weak binders respectively and W is the concentration of the weak binder 43 GE Title or job number 11/2/2012 Displacement ITC – HIV-1 Protease- Inhibitor Binding Amprenavir Acetyl pepstatin Amprenavir + acetyl pepstatin Unable to determine KB KB of 3.1 x 1010 M-1 Ohtaka, et al, Protein Sci.
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