Wei Li - PowerPoint Presentation

Slide1

Wei LiDepartment of ChemistryUniversity of VictoriaWinter, 2013

Measurement of Binding Constants and Heats of Binding using Isothermal Titration CalorimeterSlide2

Acknowledgement Professor HofClassmatesSlide3

OutlineThermodynamic parametersIsothermal titration calorimeter (ITC)ITC development historyAdvantages of using ITCBasic configuration of an ITCSample preparationRaw data and plot of processed dataSystematic errors

ReferencesSlide4

Thermodynamic Parameters When characterizing interactions between a biological macromolecule M and a small ligand X, or between two macromolecules, M + X = MX

MX + X = MX

2

…….

MX

n-1

+ X =

MXn, K = [MXn]/[MXn-1] X] G = G° + RTlnK (Standard conditions: 1 mole of P and 1 mole of L at PH7 and 25° C) At equilibrium, under standard conditions, G = 0. G° = -RTlnK = H° - TS°

The single-site binding constant K,



H°

,

and the number of sites n are the

independent variables of thermodynamic

interest.



G° and



S° of binding are dependent

variables obtained by the calculation.Slide5

Thermodynamic ParametersEntropyHydrophobic interactionsWater release Ion release Conformational changes EnthalpyHydrogen bonding Protonation events

n

= stoichiometry

Number of protein binding

sites reflects

the purity and the functional integrity of a protein preparation if the measure and fitted stoichiometry can be compared to the known, previously determined stoichiometry

Slide6

ITC Development History1960s – built in the second half to study chemical reactions1970s – sensitivity: mJ metal + ligand complex1980s – sensitivity: µJ

ligand binding processes and micelle formation

1990s – number of published papers increased due to new commercial calorimeters became available. Evolved from a specialist method to a widely used technique

2000s – widely employed in the design and discovery of new drugs and for the study of liquid mixtures

2009 – 374 out of 432 papers on protein interaction with other proteins, small molecules, metal ions, lipids, nucleic acids, and carbohydrates as well as on nucleic acid interactions with small molecules.Slide7

Advantages of using ITCNo labeling requiredIn-solutionNo molecular weight limitationsOptical clarity unimportantMinimal assay developmentUsing heat as signal

In one single experiment, K, ∆H, and Stoichiometry n of interaction between two or more molecules in solution can be determined

∆G and ∆S can be calculated

Mark A. Williams and Tina

Daviter

(

eds

), Protein-Ligand Interactions: Methods and Applications. Springer Science and Business Media, New York, 2013Slide8

Basic Configuration of an ITCReference cell and sample cell in an adiabatic jacket. Reference cell filled with buffer or water. Sample cell filled with protein.Both cells are heated in such a way that the temperature is almost constant, i.e. T < 10 x (E-6)

°

C at all times.

Syringe device holds the ligand. This device titrates the ligand into the sample cell and also acts as a stirrer.

“Isothermal titration

calorimetry

(ITC) is a biophysical technique that allows a thermodynamic characterization of an interactive system”.

VP-ITC micro calorimeterSlide9

Basic Configuration of an ITCReference and sample cells containing identical buffer are located within an adiabatic jacket, and the latter contains macromolecule of interest.A small, constant power is applied to the reference cell, which activated the cell feedback circuit to drive ∆T to 0.No reaction – the feedback power remains constant at the resting baseline value.Exothermal reactions temporarily ↓ and endothermic reactions temporarily ↑ feedback power. The reaction heats are readily obtained by computer integration of these deflections from the resting baseline. Slide10

Sample PreparationGeneral considerationExperiment performed at reasonable PHProtein has to be extensively dialysed and the ligand has to be dissolved in the buffer recovered from the last protein dialysis stepProtein preparationAs pure as possible

Concentration should be correctly estimated.

Ligand preparation

As pure as possible

Accurate concentration

Buffers

Buffers with low ionization enthalpy should be considered.Slide11

Raw Data and Plot of Processed DataTop: Raw ITC data showing an exothermic binding reaction. Each peak corresponds to an injection of ligand into a protein solution in the sample cell; the area under the peak is proportional to the amount of heat released in the binding reaction. When the protein becomes saturated, the DP signal diminishes until only the background heat of dilution is observed.Bottom: Binding isotherm obtained by integrating the area of each peak. The heat released per mole of ligant is plotted against the molar ratio of the two reactants. Slide12

Systematic ErrorsBlank experimentHeat of dilution of ligandHeat of dilution of macromoleculeBuffer to buffer experimentHeat of proton ionization

Degassing

Generation of bubbles during an ITC experiment will generate spurious heat signals.

Measurements of the concentrations

Accuracy of the measurements are imperative for a good ITC experiment.

Sample concentrations also need to be within a proper range to get reliable data.

Small molecules impurities and PH mismatches in the buffer will cause artifacts.Slide13

ReferencesNunez, S.; Venhorst, J.; Kruse, C. G. Target–drug interactions: First principles and their application to drug discovery. Drug Discov. Today 2012, 17, 10–22.Wiseman T. et al, Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal Biochem 179: 131-137, 1989.Mark A. Williams and Tina

Daviter

(

eds

), Protein-Ligand Interactions: methods and applications. Springer Science and Business Media, New York, 2013.

Joel

Tellinghuisen

, John D. Chodera, Systematic errors in isothermal titration calorimetry: concentrations and baselines, Analytical Biochemistry 414 (2011) 297-299.wikepediaSlide14

Transferrin

Human serum albuminSlide15