title: Size-exclusion chromatography can identify faster-associating protein complexes and evaluate design strategies.
abstract: 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).
  
  
  RESULTS
  Small-zone size-exclusion chromatography (SEC) detected persistent high-affinity MICA mutant-NKG2D complexes in solution as early-eluting peaks. The SEC binding assay used standard protein purification instrumentation to evaluate complex stability, qualitatively paralleled the SPR results, and successfully discriminated among complexes that differed only in on-rates. We used the SEC binding assay, along with SPR, to assess the results of a follow-up design strategy targeting the non-interfacial redesigned region. Both SEC and SPR agreed that these mutations did not enhance affinity as much as previous mutants. When the SEC binding assay was run in 1 M urea, only the highest affinity complex was detected.
  
  
  CONCLUSIONS
  This SEC binding assay provides a correlation with SPR results for protein complex affinities, detecting changes in complex on-rates, and tunable to lower sensitivity with 1 M urea. The SEC binding assay is complementary to other protein design evaluation methods, can be adapted to the undergraduate research laboratory, and may provide additional structural information about changes in hydrodynamic radii from elution times. Our assay allowed us to conclude that further alteration of MICA at non-contacting residues is unlikely to further enhance NKG2D affinity.
authors: Department of Chemistry and Biochemistry, Seattle Pacific University, 3307 Third Avenue West, Seattle, WA 98119-1997, USA. mayerc@spu.edu
authors: Chad L
authors: CL
authors: Mayer
authors: W Kalani
authors: WK
authors: Snyder
authors: Monika A
authors: MA
authors: Swietlicka
authors: Andrew D
authors: AD
authors: Vanschoiack
authors: Chad R
authors: CR
authors: Austin
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