Long-range-distance NMR effects in a protein labeled with a lanthanide-DOTA chelate

A two-thiol reactive lanthanide-DOTA (1,4,7,10-tetraazacyclododecane-N,N',N,N'-tetraacetic acid) chelate. CLaNP-3 (CLaNP = caged lanthanide NMR probe), was synthesized for the rigid attachment to cysteine groups on a protein surface, and used to obtain long-range-distance information from the {N-15, H-1} HSQC spectra of the protein-lanthanide complex. The DOTA ring exhibits several isomers that are in exchanged however single resonances were observed for most amide groups in the protein, allowing determination of a single, apparent magnetic-susceptibility tensor. Pseudocontact shifts caused by Yb-containing CLaNP-3 were observed for atoms at 15-35 angstrom from the metal. By using Gd-containing CLaNP-3, relaxation effects were observed, allowing distances up to 30 angstrom from the paramagnetic center to be determined accurately. Similar results were obtained with a Gd-DTPA (diethylene-triamine-pentaacetic acid) chelate, CLaNP-1, bound in the same bidentate manner to the protein. This study demonstrates that bidentate attachment of a paramagnetic probe enables determination of long-range distances.