Converting weak binders into infinite binders.
Monoclonal antibody 2D12.5 binds DOTA chelates of all the rare earths with K(d) approximately 10(-)(8) M, making it useful for the capture of probe molecules with a variety of properties. To make 2D12.5 even more useful for biological applications, we have engineered a single cysteine residue at position 54 of the heavy chain, a site proximal to the protein's binding site, so that weakly electrophilic metal complexes of (S)-2-(4-acrylamidobenzyl)-DOTA (AABD) may bind and form permanent linkages. At 37 degrees C, pH 7.5, all of the rare earth-AABD complexes bind permanently to the 2D12.5 G54C mutant within 5 min, in yields that correlate with their relative binding affinities. Surprisingly, indium-AABD also binds permanently in >50% yield within 5 min, despite the fact that changing the metal to indium reduces the affinity approximately 100x; even copper-AABD, which has approximately 10 000x lower binding affinity than the rare earths, binds permanently in >70% yield within 2 h. However, acrylamido compounds with no measurable affinity do not bind permanently. The important practical implication is that the G54C mutant of 2D12.5 may be used for applications that include not only the rare earths, but also an unexpected range of other elements as well. This infinite binding system can exhibit selective and permanent attachment with a remarkable range of structurally related ligands, albeit at slower rates as affinities decrease.
Corneillie
Todd M
TM
Department of Chemistry, University of California, Davis, California 95616, USA.
Whetstone
Paul A
PA
Lee
Kelvin C
KC
Wong
Jeremy P
JP
Meares
Claude F
CF
D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology; D008674:Metals, Rare Earth / Q000378:metabolism; D009154:Mutation; D015019:Yttrium / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D008674:Metals, Rare Earth; D015019:Yttrium