Exact analysis of heterotropic interactions in proteins: Characterization of cooperative ligand binding by isothermal titration calorimetry.
Intramolecular interaction networks in proteins are responsible for heterotropic ligand binding cooperativity, a biologically important, widespread phenomenon in nature (e.g., signaling transduction cascades, enzymatic cofactors, enzymatic allosteric activators or inhibitors, gene transcription, or repression). The cooperative binding of two (or more) different ligands to a macromolecule is the underlying principle. To date, heterotropic effects have been studied mainly kinetically in enzymatic systems. Until now, approximate approaches have been employed for studying equilibrium heterotropic ligand binding effects, except in two special cases in which an exact analysis was developed: independent binding (no cooperativity) and competitive binding (maximal negative cooperativity). The exact analysis and methodology for characterizing ligand binding cooperativity interactions in the general case (any degree of cooperativity) using isothermal titration calorimetry are presented in this work. Intramolecular interaction pathways within the allosteric macromolecule can be identified and characterized using this methodology. As an example, the thermodynamic characterization of the binding interaction between ferredoxin-NADP+ reductase and its three substrates, NADP+, ferredoxin, and flavodoxin, as well as the characterization of their binding cooperativity interaction, is presented.
Velazquez-Campoy
Adrian
A
Institute of Biocomputation and Complex Systems Physics (BIFI), Universidad de Zaragoza, Zaragoza, Spain. adrianvc@unizar.es
Goñi
Guillermina
G
Peregrina
Jose Ramon
JR
Medina
Milagros
M
D000465:Algorithms; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning / Q000379:methods*; D003198:Computer Simulation; D006358:Hot Temperature; D008024:Ligands; D008956:Models, Chemical; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000648:ultrastructure*; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D025941:Protein Interaction Mapping / Q000379:methods*; D017186:Titrimetry / Q000379:methods
D008024:Ligands; D046912:Multiprotein Complexes