C. Eichmuller et al., Mapping the ligand binding site at protein side-chains in protein-ligand complexes through NOE difference spectroscopy, J BIOM NMR, 20(3), 2001, pp. 195-202
This report describes a novel NMR approach for mapping the interaction surf
ace between an unlabeled ligand and a C-13,N-15-labeled protein. The method
relies on the spin inversion properties of the dipolar relaxation pathways
and records the differential relaxation of two spin modes, where ligand an
d protein H-1 magnetizations are aligned either in a parallel or anti-paral
lel manner. Selective inversion of protein protons is achieved in a straigh
tforward manner by exploiting the one-bond heteronuclear scalar couplings (
(1)J(CH), (1)J(NH)). Suppression of indirect relaxation pathways mediated b
y bulk water or rapidly exchanging protons is achieved by selective inversi
on of the water signal in the middle of the NOESY mixing period. The method
does not require deuteration of the protein or well separated spectral reg
ions for the protein and the ligand, respectively. Additionally, in contras
t to previous methods, the new experiment identifies side-chain enzyme liga
nd interactions along the intermolecular binding interface. The method is d
emonstrated with an application to the B-12-binding subunit of glutamate mu
tase from Clostridium tetanomorphum for which NMR chemical shift changes up
on B-12-nucleotide loop binding and a high-resolution solution structure ar
e available.