ABSTRACT
Biological processes are dependent on interactions of proteins with other proteins, carbohydrates, nucleic acids, lipids, or smaller molecule ligands. Two fundamental problems exist: the determination of what molecules interact with a given protein, and characterization and quanti cation of the interaction once it is known. This chapter focuses on the latter. Methodologies to quantify protein interactions with other molecules are numerous. Two methods that provide fundamental thermodynamic and kinetic parameters are calorimetry and surface plasmon resonance (SPR). There are two general calorimetric techniques used in studying biological processes: differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). DSC measures heat capacity and enthalpy during denaturation processes; ITC measures heat evolved or absorbed during molecular association. ITC has a wide range of applications and can be used to measure free energy change,
19.1 General Strategies for Characterization of Protein Interactions .......................................... 351 19.2 Isothermal Titration Calorimetry ......................................................................................... 352
19.2.1 Experimental Considerations .................................................................................... 352 19.2.1.1 Sample Preparation .................................................................................... 353 19.2.1.2 Buffer Selection ......................................................................................... 353
19.2.2 Determination of Binding Af nity and ΔH ............................................................. 353 19.2.3 Reference Titration to Determine Heat of Ligand Dilution ...................................... 355 19.2.4 Data Analysis ............................................................................................................ 355 19.2.5 Troubleshooting Guide ............................................................................................. 356
19.3 Surface Plasmon Resonance ................................................................................................. 356 19.3.1 Experimental Considerations .................................................................................... 357 19.3.2 Buffer Solutions ........................................................................................................ 357 19.3.3 Sensor Chip ............................................................................................................... 358
19.3.3.1 Immobilized Molecule ............................................................................... 359 19.3.3.2 Regeneration of Sensor Surface ................................................................. 359
19.3.4 Procedures ................................................................................................................ 359 19.3.4.1 Check for Nonspeci c Binding to Sensor Chip .........................................360 19.3.4.2 Immobilization of Capture Molecule by Primary Amine Coupling ......... 361 19.3.4.3 Binding of Ligand to Capture Molecule .................................................... 361 19.3.4.4 Binding of Analyte to Capture Ligand Molecule ...................................... 361 19.3.4.5 Kinetic Data for Protein Interactions ......................................................... 362 19.3.4.6 Data Analysis ............................................................................................. 362
19.3.5 Troubleshooting Guide ............................................................................................. 362 References ...................................................................................................................................... 363
enthalpy change, entropy change, stoichiometry of binding, and heat capacity change (Perozzo et al. 2004). SPR measures in real time changes in refractive index upon binding of a molecule to another molecule, and therefore, can be a useful technique to analyze the kinetics of protein interactions including on and off rates.
