A Quantitative Model of Thermal Stabilization and Destabilization of Proteins by Ligands

Abstract

Equilibrium binding ligands usually increase protein thermal stability by an amount proportional to the concentration and affinity of the ligand. High-throughput screening for the discovery of drug-like compounds uses an assay based on thermal stabilization. The mathematical description of this stabilization is well developed, and the method is widely applicable to the characterization of ligand-protein binding equilibrium. However, numerous cases have been experimentally observed where equilibrium binding ligands destabilize proteins, i.e., diminish protein melting temperature by an amount proportional to the concentration and affinity of the ligand. Here, we present a thermodynamic model that describes ligand binding to the native and unfolded (denatured) protein states explaining the combined stabilization and destabilization effects. The model also explains nonsaturation and saturation effects on the protein melting temperature when the ligand concentration significantly exceeds the protein concentration. Several examples of the applicability of the model are presented, including specific sulfonamide binding to recombinant hCAII, peptide and ANS binding to the Polo-box domain of Plk1, and zinc ion binding to the recombinant porcine growth hormone. The same ligands may stabilize and destabilize different proteins, and the same proteins may be stabilized and destabilized by different ligands.

Sensor Domain of Histidine Kinase KinB of Pseudomonas

“…However, binding of a signal molecule may disrupt an SD dimer or lower its stability. Both protein thermal stabilization and destabilization by bound ligands have been observed and examined (56). The binding of monophosphate sugars to the KinB-SD may be an example of the latter case, as observed in our DLS experiments.…”

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

“…Our experience has been that careful consideration of all likely factors in the experiment at the stage of taking initial readings is essential to obtaining the best results. Furthermore, alternative theoretical treatments can reveal features of these data 15,17 . Finally, it is not uncommon for some proteins that contain natively exposed hydrophobic regions to show high background fluorescence.…”

Benzenesulfonamides with benzimidazole moieties as inhibitors of carbonic anhydrases I, II, VII, XII and XIII

“…Expression and purification of CA I, II, VII, XII and XIII has been previously described: CA I by Baranauskiene et al 28 , CA II by Cimmperman et al 29 , CA VII and XIII by Sudzius et al 21 and CA XII by Jogaite et al 30 .…”