Mapping allostery through equilibrium perturbation NMR spectroscopy.
The understanding of allostery relies on the comparative analysis of macromolecules in their free and bound states. However, the direct free versus bound comparison is often challenging due to the instability of one of the two forms. This problem is effectively circumvented by using minor free/bound equilibrium perturbations which are tolerated without compromising sample stability. The subtle equilibrium perturbations are still able to reveal significant apo/holo differences if monitored by NMR experiments that are sensitive to minor populations within dynamic equilibria, such as NMR relaxation dispersion (NMRD) and hydrogen exchange (H/D and H/H) rates. These measurements are complementary to each other as they unmask how a ligand affects both the stable and the excited states of the free energy landscape for its protein receptor. The proposed equilibrium perturbation approach therefore significantly expands the scope of applicability of NMRD and hydrogen exchange experiments to the investigation of ligand-protein interactions, in general, unveiling allosteric "hot spot" maps for systems that have been traditionally elusive to direct free/bound comparisons.
Das
Rahul
R
Department of Biochemistry and Biomedical Sciences, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4M1, Canada.
Abu-Abed
Mona
M
Melacini
Giuseppe
G
D000494:Allosteric Regulation; D001665:Binding Sites; D003903:Deuterium / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D012680:Sensitivity and Specificity
D008024:Ligands; D006859:Hydrogen; D003903:Deuterium