Label-free assay for thermodynamic analysis of protein-ligand interactions: a multivariate strategy for allosteric ligand screening.
The binding of allosteric ligands to protein can induce changes to the holoprotein conformation, stability, and activity that have an impact on unfolding dynamics. Herein we report a label-free strategy for determining the dissociation constant of protein-ligand interactions over a wide dynamic range (>10(4), Kd from nano- to millimolar) using capillary electrophoresis that overcomes the constraints of an ideal two-state protein unfolding model. Multivariate analysis of thermodynamic parameters associated with holoprotein unfolding and ligand binding is demonstrated for the classification of cyclic nucleotide analogues that function as allosteric modulators of regulatory proteins, such as the exchange protein directly activated by cAMP (EPAC).
Gavina
Jennilee M A
JM
Department of Chemistry, McMaster University, 1280 Main Street West, Hamilton, ON, Canada L8S 4M1.
Mazhab-Jafari
Mohammad T
MT
Melacini
Giuseppe
G
Britz-McKibbin
Philip
P
D000494:Allosteric Regulation; D001681:Biological Assay; D055592:Biophysical Phenomena; D019075:Electrophoresis, Capillary / Q000379:methods; D007700:Kinetics; D008024:Ligands; D015999:Multivariate Analysis; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011506:Proteins / Q000378:metabolism*; D013816:Thermodynamics
D008024:Ligands; D011506:Proteins