title: Binding techniques to study the allosteric energy cycle.
abstract: Thermodynamic principles of cooperativity and allostery have long been used as a starting point to begin understanding the interplay between ligand binding events. Understanding the nature of allosteric effects requires an experimental technique that can be used to quantify ligand binding energies and simultaneously give experimental insights into the conformational dynamics at play upon ligand binding. CD spectroscopy provides macroscopic information about the relative secondary and tertiary structures present in a protein. Here, we use this spectroscopic technique with thermal shift assays wherein ligand binding constants can be quantified based on their stabilizing effect against thermally induced protein denaturation. Binding constants for two ligands are used to determine a pairwise coupling free energy which defines the shared energy that favors or opposes binding of the second ligand binding event in an allosteric system. In CD-based thermal shift assays, temperature is the driving force for protein unfolding and can also influence protein conformational dynamics present in the unbound protein or ligand-bound proteins. Dihydrofolate reductase (DHFR) and glutamate dehydrogenase (GDH) are proposed as example test systems. NADP and methotrexate bind DHFR with positive cooperativity. Mammalian GDH exhibits negative cooperativity with respect to binding of NAD and NADPH coenzyme molecules, activation by ADP, and inhibition by GTP.
authors: Biopharmaceuticals Research & Development, GlaxoSmithKline, Upper Merion, PA, USA. james.k.kranz@gsk.com
authors: James K
authors: JK
authors: Kranz
authors: José C
authors: JC
authors: Clemente
mesh_terms: D000494:Allosteric Regulation / Q000502:physiology; D002942:Circular Dichroism; D005969:Glutamate Dehydrogenase / Q000378:metabolism; D008024:Ligands; D011485:Protein Binding; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D008024:Ligands; D005969:Glutamate Dehydrogenase; D013762:Tetrahydrofolate Dehydrogenase