ASAP J. Am. Chem. Soc., ASAP Article, 10.1021/ja805839y

Dynamics of Ligand Binding from 13C NMR Relaxation Dispersion at Natural Abundance

John S. Zintsmaster, Brian D. Wilson, and Jeffrey W. Peng*

Abstract:
We show that Carr−Purcell−Meiboom−Gill (CPMG) 13Cα NMR relaxation dispersion measurements are a viable means for profiling μs−ms ligand dynamics involved in receptor binding. Critically, the dispersion is at natural 13C abundance; this matches typical pharmaceutical research settings in which ligand isotope-labeling is often impractical. The dispersion reveals ligand 13Cα nuclei that experience μs−ms modulation of their chemical shifts due to binding. 13Cα shifts are dominated by local torsion angles φ, ψ, χ1; hence, these experiments identify flexible torsion angles that may assist complex formation. Since the experiments detect the ligand, they are viable even in the absence of a receptor structure. The μs−ms dynamic information gained helps establish flexibility−activity relationships. We apply these experiments to study the binding of a phospho-peptide substrate ligand to the peptidyl-prolyl isomerase Pin1.