Evaluation of protein adsorption and preferred binding regions in multimodal chromatography using NMR.
NMR titration experiments with labeled human ubiquitin were employed in concert with chromatographic data obtained with a library of ubiquitin mutants to study the nature of protein adsorption in multimodal (MM) chromatography. The elution order of the mutants on the MM resin was significantly different from that obtained by ion-exchange chromatography. Further, the chromatographic results with the protein library indicated that mutations in a defined region induced greater changes in protein affinity to the solid support. Chemical shift mapping and determination of dissociation constants from NMR titration experiments with the MM ligand and isotopically enriched ubiquitin were used to determine and rank the relative binding affinities of interaction sites on the protein surface. The results with NMR confirmed that the protein possessed a distinct preferred binding region for the MM ligand in agreement with the chromatographic results. Finally, coarse-grained ligand docking simulations were employed to study the modes of interaction between the MM ligand and ubiquitin. The use of NMR titration experiments in concert with chromatographic data obtained with protein libraries represents a previously undescribed approach for elucidating the structural basis of protein binding affinity in MM chromatographic systems.
Chung
Wai Keen
WK
Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
Freed
Alexander S
AS
Holstein
Melissa A
MA
McCallum
Scott A
SA
Cramer
Steven M
SM
D000327:Adsorption; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006801:Humans; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics
D011506:Proteins; D025801:Ubiquitin