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Analysis of Macromolecular Interactions by Isothermal Titration Calorimetry

Analysis of Macromolecular Interactions by Isothermal Titration Calorimetry A primer on the experimental thermodynamic charaterisation of binding of biomolecules. Gonzalo Obal Protein Biophysics Unit Structural Biology Platform Institut Pasteur de Montevideo.

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Analysis of Macromolecular Interactions by Isothermal Titration Calorimetry

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  1. Analysis of Macromolecular Interactions by Isothermal Titration Calorimetry A primer on the experimental thermodynamic charaterisation of binding of biomolecules Gonzalo Obal Protein Biophysics Unit Structural Biology Platform Institut Pasteur de Montevideo

  2. Biomolecular interactions: why are they important? Corpora non agunt nisi ligata Paul Ehrlich All (or almost all) biological phenomena depends on interactions between molecules Antibody-antigen / Enzyme-sustrate / Receptor-hormone / Signaling cascades Protein-lipid / Protein-carbohydrate / Protein-peptide / Protein-DNA Protein-RNA / ADN-ARN /…etc, etc,etc….. Binding of macromolecules is the basis of molecular especificity (ie discrimination between partners and non-partners So, without binding there is no biology

  3. Protein-protein interaction network map in yeast. Shown are the 2358 known interactions for the 1548 proteins actually available in the yeast proteome. Color correlates with function: membrane fusion (blue), cromatin structure (grey), celular structure (green), lipid metabolism (yellow), cytoquinesis (red).Schwikowski, Uetz & Fields (2000) A network of interacting proteins in yeast. Nat. Biotechnol. 18, 1257–1261

  4. A remarkable example: metal sensing E.coli • Thevolume of anE. colicellisabout 1.8E-15 L... • …thus, thelowest intracelular Zn2+croncentrationisabout 1E-9M • (i.e. 1 ion Zn2+ per bacteria!). • Zn2+ sensor in E. colimanageuptake/expulsion of ions, being sensible toconcentrationbelow10E-15M … • …thus, in tipicalconditions, intracelular concentration of Zn2+shouldbe of lessthan 1 átomo Zn2+/E. coli Femtomolar Sensitivity of Metalloregulatory Proteins Controlling Zinc Homeostasis Outten & O’Halloran (2001) Science 292, 5526 • Thus...a complete interpretation of any interaction under a particular biological scenario requires the knowledge of both the strength of binding and concentration of the molecules involved • So, consideritwhenstudyingbinding: • Thebiologicalrelevance of Kddepensonthe actual concentrations of interactingpartners • Free protein and ligandconcentrationswilldictatetheextent of binding, viaKd • Thermodynamic and kinetic control of reactionsexists…..´´even´´ in biology

  5. The goals of binding studies are to aswer the questions: HOW MANY? HOW TIGHTLY? HOW FAST? WHERE? WHY? HOW? Bindingstudiesshouldultimatelyelucidateeachquestion in ordertoprovide a complete understanding of a biomolecularinteraction…… (thisis a long, complex, interdisciplinarytask)

  6. What information do we need to get a full characterisation of an interaction?(from a biophysical point of view) THERMODYNAMICS Stoichimetry Affinity (strength) otherthermodynamicproperties (H, S, Cp) KINETICS rateconstantsforassociation (kon) and disociation (koff) Reactionmechanism STRUCTURE Three-dimensional structure of the individual interacting partners and their complex(es) DYNAMICS Molecular dynamics of individual interacting partners and their complex(es)

  7. How to analyse protein-ligand interations?There´s a wide toolkit of methods (with both advantages and pitfalls).. • … qualitative methodologies (yes/no information)… • Double-hybrid • Pull-down method (TAP, co-precipitation) in vivo e in vitro • protein arrays • …and quantitatives • Equilibrium dialysis • EMSA / native PAGE / Blue Native Page • ELISA / RIA • Fluorescence (FRET, quenching, anisotropy) • Light scattering • Surface plasmon resonance • Surface wave resonance • Asymmetric flow-filled fractionation • Stopped-flow (coupled to a wide option of detectors) • ITC • NMR • AUC • QCM (quartz crystal microbalance/piezoelectric acoustic sensor

  8. The basics of binding interactions Definition of binding affinity for macromolecular recognition The binding of two (any number) of proteins can be viewed as a reversible process, in an equilibrium governed by the law of mass action

  9. The (very) basics of binding interactions Definition of binding affinity for macromolecular recognition

  10. General properties of binding isotherms: Fractional Saturation Y = [X]/([X]+Kd) ForX = 0 Y = 0 nothing bound ForX   Y = 1 full occupancy For X = KD Y = 0.5 half occupancy

  11. Thermodynamicproperties of a bindingreaction Bindingconstantsprovideanentryintothermodynamics…and viceversa

  12. Thermodynamicproperties of a bindingreaction Bindingconstantsprovideanentryintothermodynamics…and viceversa

  13. To analyse a binding reaction we need somehow to ´´see´´ how substrates are being consumed and/or products are being formed • So, we´ll follow the extent of the reaction KA  MX /M X 1/KD Complex formed unbound macromolecule unbound ligand Basically, the idea of every binding experiment involves fixing the concentration of one of the interactors (tipically M) and varying the one of the other, having found some ´´signal´´ that changes proportionally to the amount of complex formed.

  14. ...so we need some signal for monitoring a binding reaction (i.e. follow/determine Y) …-direct measurement of the concentration of one interactor-fluorescence (intrinsic/extrinsic-anisotropy-FRET-heat-………… ¿which one and how do we use it? • Two general methodsfordeterminingbindingconstants: • Measurebound vs. Free ligand/protein at equilibium as a function of concentration • Measureassociation and dissociationrateconstants and use thesetocalculatebindingconstants

  15. How do wecalculateaffinityfrom a titrationexperiment? so…we ``follow´´ a signal proportional to the amount of complex formed as a function of x where: MX,M, y X are the concentrations for free complex, macromolecule, and ligand First: Total macromolecule and ligand concentration are: So we write Kd as: Rearranging terms: As: We re-write: O en su forma mas común:

  16. WORKFLOW OF QUANTITATIVE CHARACTERISATION OF BIOMOLECULAR BINDING INTERACTIONS PRODUCTION OF REACTANTS CHARACTERISATION OF REACTANTS SELECTION OF METHOD(S) IMPROVED EXPERIMENTAL DESIGN SIMULATION BINDING EXPERIMENT INTERPRETATION (BIOLOGICAL SIGNIFICANCE) Equilibrium MODEL SELECTION and/or CONSTRUCTION RESULTS DATA ANALYSIS (FITTING & STATISTICS) BINDING EQUATIONS

  17. Isothermal Titration Calorimetry Overview and Applications Theory and Instrumentation Experimental setup Concepts of ITC data analysis Examples: interpreting ITC results in context of structural thermodynamics

  18. Isothermal Titration Calorimetry • ITC measures the heat uptake or release during a biomolecular reaction • Heat is taken up (absorbed, endothemic) • Heat is evolved (released, exthermic) • Calorimetry is the only method that can directly measure the binding energetics of biological processes

  19. Microcalorimetry provides binding stoichiometry • Number of ligand binding sites per macromolecule – on a molar basis, model-independent (by convention a ´´Ligand´´ has one binding site, and a ´´Macromolecule´´ can have more than one)

  20. Microcalorimetry is a gold-standard for binding analysis • Label-free • In-solution • No MW limitations • Optical transparency/clarity unimportant • Minimal assay development

  21. Microcalorimetry is (almost) universally applicable to study interactions between: • Protein-small molecule • Enzyme-inhibitor • Protein-protein • Protein-DNA • Protein-RNA • Protein-lipid/liposomes • Protein-carbohydrate • Other non-biological binding reactions • Oligomerisation

  22. Microcalorimetryprovides a total picture of bindingenergetics (however, itisnoteasyto relate physicochemicalproperties (G, H, S) specifically and directlytobindingmechanisms) Verybroadly: i) H reflectsenergychangesassociatedwithmaking (-H) and breaking (+ H) of bonds (hidrogen , van der Wallsbonding, solvent) Entalphy-drivenreactions (highaffinity) ii) S reflectschangesassociatedwithincreasing (+ S) ordecreasing (- S) thenumber of microscopicconfigurations (hydrophobicinteractions, flexibility, solvent) Entropy-drivenreactions (highspecificity)

  23. Importantly, the relative contribution of H and S to G govern/distinguish the functional characteristics of R-L interactions Thisis a majorconcern in drugdiscovery and agonist/antagonistmechanism

  24. ITC: instrumental concepts The idea is to titrate a macromolecule (in the cell) by serially injecting a ligand (in the syringe), while recording the evolved (absorved/release) heat SampleCell Ligand X in syringe ReferenceCell Receptor M in cell T = 0

  25. VP-ITC (GE, Microcal)at UBP-BS/IPMON

  26. Stepbystep……¿how do wegetinformationfroman ITC experiment? 1.- titration: First, weperform ´´data collection: power (cal o J/seg) as a function of time (s) Everybindingevent (injection) isassociatedto a givenamount of heatreleased (exothermic) orabsorved (endothermic) P = dQ/dt

  27. …recapitulating, the concept is: In eachinjectionweaddn moles of X to a fixedamount of M (MT), releasingorabsorvingsomeheatqi Thus… at everyXT /MT, wedirectlymeasureitsassociatedqi… integration

  28. Finally… (WisemanIsotherm) …given qi for every injection we have: qi = Qreacción …experiment is done a constant T and P, thus: Qreacción = H 1 n 1 2 (Ajuste a modelo n sitios de unión) 3 4

  29. Theshape of bindingisothermsdepends of thevallue of KD (thisisimportantfrom a practicalconsideration, as thislimitstheconditions of theexperiment)

  30. Data analysis i) Global ITC data analysis Global fitting routines to different binding models Situation could complicate for n2 Cooperativity is sometimes difficult to analyse (specially negative coopeative) ii) Optimisation and Statistical error analysis Monte Carlo simulations of binding data Variance-Covariance Matricial analysis of statistical significance iii) Global Multimethod Analysis (GMMA) Sometimes ITC (or any other technique) data are not sufficient for obtaning a plausible explanation Combination of other methodologies are required (orthogonal information)

  31. Other complication: proteins (biomolecules) are dynamics (native ensemble)

  32. Other complication: proteins (biomolecules) are dynamics (native ensemble) Induced fit Conformational selection Conformational selection + Induced fit

  33. cAMPbindingtocataboliteinhibitoryprotein CAP CAP isanhomodimericprotein whatdoesthe ITC shows? Suggeststwosites (independent? sequential?) …wherebinding of a secondcAMPmoleculesisentropicallydisfavourable

  34. How can we explain negative cooperativity from the thermodynamic properties of? Checkforentropychange (conformational) of theproteinduringcAMPbinding… S S

  35. Thus, a thermodynamicmodel can bepropose: Binding of 1 cAMP: Boundmonomerrigidifies Binding of 2 AMP: Allpoteinrigidifies No cAMP: Bothmonomers are flexible AffinityfocAMPisidentical at bothsites

  36. Thanks!Questions?

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