Size-exclusion chromatography can identify faster-associating protein complexes and evaluate design strategies.

Background: We previously developed a set of rationally designed mutant MICA protein ligands for the NKG2D immunoreceptor in which MICA was mutated at residues that do not contact NKG2D. Some of these MICA mutants, predicted by RosettaDesign to be destabilized, bound NKG2D with affinities enhanced by more than an order of magnitude when evaluated by surface plasmon resonance (SPR).

Findings: Small-zone size-exclusion chromatography (SEC) detected persistent high-affinity MICA mutant-NKG2D complexes in solution as early-eluting peaks.