Molecular Interactions @ BIFI ITC & SPR

1. Molecular Interactions @ BIFI ITC & SPR Adrián Velázquez Campoy

3. Molecular interaction techniques Dialysis Ultrafiltration Ultracentrifugation (sedimentation equilibrium and velocity) Chromatography (size exclusion, affinity) Membrane-filter binding Capillary electrophoresis Gel-shift electrophoresis UV/Visible spectroscopy Fluorescence spectroscopy (correlation, intensity, polarization, anisotropy, FRET) Circular dichroism Dynamic light scattering Nuclear magnetic resonance (HSQC, STD) IR spectroscopy Raman spectroscopy Electrochemistry Isothermal titration calorimetry Differential scanning calorimetry Surface plasmon resonance Hydroxyl radical foot-printing Protease-digestion protection Mass spectrometry Atomic force microscopy X-ray diffraction X-ray absorption fine structure Electron microscopy Biological activity (e.g. enzymatic reaction) Chemical cross-linking Two-hybrid systems Co-precipitation Western analysis Fluorescence microscopy (correlation, FRET) Flow citometry (FRET) Confocal microscopy

4. Is there interaction between two biomolecules? Yes/No What is the stoichiometry? n How strong is the interaction? G, Ka How fast does the interaction occurs? kon, koff What intermolecular forces are involved? H, -TS, CP, +cond. Is binding coupled to another binding process? nX, +molecules Is binding coupled to a conformational change? H, -TS, CP What functional groups are involved? +mutations What is the interaction specificity? +mutations

5. Characterization of ligand binding Dissection of binding energetics Characterization of ligand specificity Coupling between ligand events (homo- and heterotropic) Allosteric control of protein function (homo- and heterotropic) Ligand binding optimization Drug development and design Protein engineering and function redesign

6. Isothermal Titration Calorimetry

7. dQ/dt R S TR T=TS-TR Additional heat provided or subtracted during the thermal event in order to ensure T=0

9. Ka H n

10. Experimental considerations: Thermostatization Equilibrium (absence of kinetic effects) Physiological/stabilizing/informative conditions Solvent composition (co-solutes/co-solvents) Purity of reactants (chemical and conformational) Everything gives a heat signal Direct and reverse titrations Calibration (electrical or chemical) Concentrations: (rule of thumb...) [M]0 = 5 – 20 M 2 ml [L]0 = (10 –20)  n  [M]0 0.5 ml

11. Bovine Pancreatic Ribonuclease A 2’CMP Ka 2.9·106 M-1 H -19.3 kcal/mol n 1.02

12. Soybean Trypsin Inhibitor Pancreatic Porcine Trypsin Ka 1.5·106 M-1 H 8.4 kcal/mol n 1.2

15. FAD Synthetase ADP 0 mM MgCl2 0 mM ADP 0.5 mM MgCl2 0 mM ADP 0.5 mM MgCl2 10 mM ADP 0 mM MgCl2 10 mM Frago et al. (2009). Journal of Biological Chemistry284 6610-6619

16. cAMP Receptor Protein + cAMP 1 5.5·104 M-1 H1 -1.9 kcal/mol 2 4.2·109 M-2 H2 9.9 kcal/mol 42/12 5.4 nHill 1.40 M ML2 ML Gorshkova et al.(1995). Journal of Biological Chemistry270 21679-21683

17. HIV-1 Protease -440 cal/Kmol -350 cal/Kmol Ohtaka et al. (2002). Protein Science11 1908-1916

18. HIV-1 Protease nH = 0.02 H0 = 6.9 kcal/mol nH = 0.39 H0 = 12.0 kcal/mol Ohtaka et al. (2002). Protein Science11 1908-1916

19. KNI-272 HIV-1 PR WT nH = -0.7 H0 = -2.5 kcal/mol HIV-1 PR V82F/I84V HIV-1 Protease nH = -0.09 H0 = -4.7 kcal/mol KNI-529 KNI-272 H = -6.3 kcal/mol pKaF = 6.0 pKaC = 6.6 pKaF = 4.8 pKaC = 2.9 Velazquez-Campoy et al. (2000). Protein Science9 1801-1809

20. Human Fibroblast Growth Factor Heparin Guzman-Casado et al. (2002). International Journal of Biological Macromolecules31 45-54

21. Kd 53M Hd 5.5 kcal/mol Bovine -Chymotrypsin Burrows et al. (1994). Biochemistry33 12741-12745 Velazquez-Campoy et al. (2004). Methods in Molecular Biology261 35-54 Belo et al. (2008). Proteins70 1475-1487

22. Tetratricopeptide Repeat Domain (PP5) + MEEVD TPR G83N TPR WT TPR WT TPR G83N -TS -TS G G H H Cliff et al. (2005). Journal of Molecular Biology346 717-732

23. ITC: Advantages • Complete thermodynamic characterization: H, Ka, n, G, and S • Direct determination of the binding enthalpy (with no additional assumptions or models; no van’t Hoff enthalpy determination) • Universal signal (heat), and high sensitivity (Q ~cal) • Absence of reporter labels (chromophores, fluorophores, etc.) • Highly reproducible, and user-friendly with low maintenance cost • Non-destructive technique (sample recovery) • Interaction in solution (no need for immobilization) • Experiments with unusual systems (e.g. dispersions, intact cells) • Relatively fast and automatized technique (< 30 min/experiment)

24. ITC: Disadvantages • Signal depends on H and concentrations. What if H is close to zero? • What if affinity is very high or very low? • Heat is a universal signal, so what are we observing in the cell? • Need for additional experiments and control assays • Relatively fast and user-friendly, but no high-throughput • Very informative, but it consumes a big amount of sample • Not often used for kinetics assays • Slow binding processes may be overlooked

25. Surface Plasmon Resonance

29. ligand O O N O ligand EDC/NHS O C ligand ligand N S S NH NH NH OH OH NH2 SH S S O O O O O C C C C C O O N O PDEA EDC/NHS O C

30. RUss koff kon

32. SPR: Advantages • No complete, but reasonable thermodynamic characterization: KA, n, G • Universal signal (resonance units, RU), and high sensitivity • Absence of reporter labels (chromophores, fluorophores, etc.) • Need for very little sample • Non-destructive technique (sample recovery) • Exceptional for kinetic assays, also appropriate for equilibrium binding • Experiments with unusual systems (e.g. dispersions, intact cells) • Appropriate for high-throughput

33. SPR: Disadvantages • Indirect determination of the binding enthalpy (with additional assumptions or models; van’t Hoff enthalpy determination) • Signal depends on MW. What if analyte MW is very low? • What if affinity is very high or very low? • What if unspecific binding? Or improper immobilization? • Very informative, but it requires numerous assays • Often complaints regarding low reproducibility • Often not user-friendly with high maintenance cost