Isothermal Titration Calorimetry in Enzymology: Techniques and Applications provides a thorough, practical overview of ITC as a productive and powerful tool for quantifying enzyme catalysis and interactions with substrates, products, cofactors, and inhibitors. Here, global experts in the methodology discuss the full capabilities of ITC and how it may be applied in research, medicine, and industry and offer a basis to continue leveraging ITC as its uses expand and evolve. This book bridges the gap between research and application by giving extensive technical details on cutting edge ITC techniques across a wide swath of both ITC kinetics measurements and ITC thermodynamic measurements, alongside case studies illustrating the depth of knowledge that can be obtained. Applications discussed include measuring binding kinetics by ITC, analyzing ITC kinetic data, in vivo enzyme kinetics by ITC, analyzing enzyme kinetics in crowded solutions, studying catalysis of biological substrates, interpreting ITC binding curves, ITC in drug discovery, ITC in the development of cancer therapeutics, ITC analysis of aminoglycoside resistance enzymes, studying enzyme interactions with toxic metals, using ITC to study ubiquitination, and ITC studies of glycoside hydrolases, among others.
1. 1 Introduction - p. 3 2. 2 The instrument response time(s) - p. 4 3. 3 Experimental example of signal deconvolution with one response time - p. 6 4. 4 The different methods to obtain kinetic information - p. 8 5. 5 Quality of the response time obtained by fitting the ETC - p. 9 6. 6 Theoretical and practical considerations about injection curve fitting - p. 12 7. 7 Comparison of full kinITC with one and two response times - p. 13 8. Appendix - p. 17 9. Chapter 2. Characterizing enzyme kinetics by 2D-ITC - p. 23 10. Chapter 3. Using isothermal titration calorimetry to measure enzyme stability - p. 41 11. Chapter 4. Analysis of ITC enzyme kinetics data using the Lambert W function - p. 51 12. Chapter 5. Design and operation of small-volume isothermal titration calorimeters (ITCs) - p. 63 13. Part II. ITC thermodynamic measurements - p. P2 14. Chapter 6. The binding polynomial: A powerful tool for modeling, analyzing, and interpreting isothermal titration calorimetry assays - p. 75 15. 1 Introduction - p. 75 16. 2 The partition function: A not-so-distant concept - p. 77 17. 3 The binding polynomial - p. 82 18. 4 Properties of the binding polynomial - p. 84 19. 5 The binding equations: Chemical equilibrium and mass conservation - p. 87 20. 6 Isothermal titration calorimetry vs. other binding techniques - p. 89 21. 7 The simplest case: One ligand binding site - p. 91 22. 8 A more complex case: Two binding sites - p. 92 23. 9 Other cases (I): Conformational heterogeneity - p. 101 24. 10 Other cases (II): Heterotropic interactions - p. 105 25. 11 Conclusions - p. 112 26. Funding - p. 114 27. Acknowledgments - p. 114 28. Chapter 7. Unbiased baseline determination, global analysis, and multimethod analysis of titration isotherms in SEDPHAT and NITPIC - p. 119 29. Chapter 8. Characterization of slow-binding inhibition by isothermal titration calorimetry: The case of urease, a nickel-dependent enzyme - p. 141 30. Chapter 9. Dissecting complex binding equilibria involving protein–protein and protein–metal interactions - p. 157 31. Chapter 10. Thermodynamics of transition state analogs - p. 173 32. Chapter 11. Application of isothermal titration calorimetry in drug discovery and development - p. 215 33. Chapter 12. Insight on metalloenzymes from isothermal titration calorimetry measurements - p. 233 34. Chapter 13. Thermophilicity, substrate promiscuity, and solvent effects on thermodynamics of ligand–protein interactions studied by isothermal titration calorimetry - p. 255 35. Chapter 14. Isothermal titration calorimetry: A thermodynamic window into natural product binding with cancer targets - p. 275 36. Chapter 15. ITC of intrinsically disordered proteins - p. 291 37. Index - p. 329
Isothermal Titration Calorimetry in Enzymology: Techniques and Applications provides a thorough, practical overview of ITC as a productive and powerful tool for quantifying enzyme catalysis and interactions with substrates, products, cofactors, and inhibitors. Here, global experts in the methodology discuss the full capabilities of ITC and how it may be applied in research, medicine, and industry and offer a basis to continue leveraging ITC as its uses expand and evolve. This book bridges the gap between research and application by giving extensive technical details on cutting edge ITC techniques across a wide swath of both ITC kinetics measurements and ITC thermodynamic measurements, alongside case studies illustrating the depth of knowledge that can be obtained. Applications discussed include measuring binding kinetics by ITC, analyzing ITC kinetic data, in vivo enzyme kinetics by ITC, analyzing enzyme kinetics in crowded solutions, studying catalysis of biological substrates, interpreting ITC binding curves, ITC in drug discovery, ITC in the development of cancer therapeutics, ITC analysis of aminoglycoside resistance enzymes, studying enzyme interactions with toxic metals, using ITC to study ubiquitination, and ITC studies of glycoside hydrolases, among others.
1. 1 Introduction - p. 3 2. 2 The instrument response time(s) - p. 4 3. 3 Experimental example of signal deconvolution with one response time - p. 6 4. 4 The different methods to obtain kinetic information - p. 8 5. 5 Quality of the response time obtained by fitting the ETC - p. 9 6. 6 Theoretical and practical considerations about injection curve fitting - p. 12 7. 7 Comparison of full kinITC with one and two response times - p. 13 8. Appendix - p. 17 9. Chapter 2. Characterizing enzyme kinetics by 2D-ITC - p. 23 10. Chapter 3. Using isothermal titration calorimetry to measure enzyme stability - p. 41 11. Chapter 4. Analysis of ITC enzyme kinetics data using the Lambert W function - p. 51 12. Chapter 5. Design and operation of small-volume isothermal titration calorimeters (ITCs) - p. 63 13. Part II. ITC thermodynamic measurements - p. P2 14. Chapter 6. The binding polynomial: A powerful tool for modeling, analyzing, and interpreting isothermal titration calorimetry assays - p. 75 15. 1 Introduction - p. 75 16. 2 The partition function: A not-so-distant concept - p. 77 17. 3 The binding polynomial - p. 82 18. 4 Properties of the binding polynomial - p. 84 19. 5 The binding equations: Chemical equilibrium and mass conservation - p. 87 20. 6 Isothermal titration calorimetry vs. other binding techniques - p. 89 21. 7 The simplest case: One ligand binding site - p. 91 22. 8 A more complex case: Two binding sites - p. 92 23. 9 Other cases (I): Conformational heterogeneity - p. 101 24. 10 Other cases (II): Heterotropic interactions - p. 105 25. 11 Conclusions - p. 112 26. Funding - p. 114 27. Acknowledgments - p. 114 28. Chapter 7. Unbiased baseline determination, global analysis, and multimethod analysis of titration isotherms in SEDPHAT and NITPIC - p. 119 29. Chapter 8. Characterization of slow-binding inhibition by isothermal titration calorimetry: The case of urease, a nickel-dependent enzyme - p. 141 30. Chapter 9. Dissecting complex binding equilibria involving protein–protein and protein–metal interactions - p. 157 31. Chapter 10. Thermodynamics of transition state analogs - p. 173 32. Chapter 11. Application of isothermal titration calorimetry in drug discovery and development - p. 215 33. Chapter 12. Insight on metalloenzymes from isothermal titration calorimetry measurements - p. 233 34. Chapter 13. Thermophilicity, substrate promiscuity, and solvent effects on thermodynamics of ligand–protein interactions studied by isothermal titration calorimetry - p. 255 35. Chapter 14. Isothermal titration calorimetry: A thermodynamic window into natural product binding with cancer targets - p. 275 36. Chapter 15. ITC of intrinsically disordered proteins - p. 291 37. Index - p. 329
