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, K a How fast does the interaction occurs? k on, k off What intermolecular forces are involved?  H, -T  S,  C P, +cond. Is binding coupled to another binding process? n X, +molecules Is binding coupled to a conformational change?  H, -T  S,  C P 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. RS  T=T S -T R dQ/dt TRTR Additional heat provided or subtracted during the thermal event in order to ensure  T=0

9. HH n KaKa

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  M2 ml [L] 0 = (10 – 20)  n  [M] 0 0.5 ml

11. Bovine Pancreatic Ribonuclease A 2’CMP K a 2.9·10 6 M -1  H -19.3 kcal/mol n 1.02

12. Soybean Trypsin Inhibitor Pancreatic Porcine Trypsin K a 1.5·10 6 M -1  H 8.4 kcal/mol n 1.2

14. K a (M -1 )  H (kcal/mol) 2’CMP2.9·10 6 M -1 -19.3 3’CMP2.4·10 5 M -1 -18.6 5’CMP4.2·10 3 M -1 -16.3

15. ADP 0 mM MgCl 2 0 mM ADP 0.5 mM MgCl 2 0 mM ADP 0 mM MgCl 2 10 mM ADP 0.5 mM MgCl 2 10 mM FAD Synthetase Frago et al. (2009). Journal of Biological Chemistry 284 6610-6619

16.  1 5.5·10 4 M -1  H 1 -1.9 kcal/mol  2 4.2·10 9 M -2  H 2 9.9 kcal/mol 4  2 /  1 2 5.4 n Hill 1.40 cAMP Receptor Protein + cAMP Gorshkova et al. (1995). Journal of Biological Chemistry 270 21679-21683 ML MML 2

17. -440 cal/K  mol -350 cal/K  mol Ohtaka et al. (2002). Protein Science 11 1908-1916 HIV-1 Protease

18. n H = 0.02  H 0 = 6.9 kcal/mol n H = 0.39  H 0 = 12.0 kcal/mol Ohtaka et al. (2002). Protein Science 11 1908-1916 HIV-1 Protease

19.  H = -6.3 kcal/mol pK a F = 6.0 pK a C = 6.6 pK a F = 4.8 pK a C = 2.9 Velazquez-Campoy et al. (2000). Protein Science 9 1801-1809 n H = -0.7  H 0 = -2.5 kcal/mol n H = -0.09  H 0 = -4.7 kcal/mol HIV-1 PR WT HIV-1 PR V82F/I84V KNI-529 KNI-272 HIV-1 Protease

20. Guzman-Casado et al. (2002). International Journal of Biological Macromolecules 31 45-54 Human Fibroblast Growth Factor Heparin

21. Burrows et al. (1994). Biochemistry 33 12741-12745 Velazquez-Campoy et al. (2004). Methods in Molecular Biology 261 35-54 Belo et al. (2008). Proteins 70 1475-1487 K d 53  M  H d 5.5 kcal/mol Bovine  -Chymotrypsin

22. Cliff et al. (2005). Journal of Molecular Biology 346 717-732 Tetratricopeptide Repeat Domain (PP5) + MEEVD TPR WT TPR G83N TPR WT TPR G83N GG -T  S HH GG HH

23. ITC:Advantages Complete thermodynamic characterization:  H, K a, 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. C OH OC O OC NH O EDC/NHS N OO NH 2 ligand C OH OC O OC NH O EDC/NHS N OO SH ligand PDEA C NH O S S S S N

30. k on k off RU ss

32. SPR:Advantages No complete, but reasonable thermodynamic characterization: K A, 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