Physicochemical and structural implications for molecular recognition in immobilized metal affinity chromatography

Abstract

A precise understanding of the mechanisms that govern selectivity in protein adsorption to metal-chelate complexes is required to provide a rational framework for predicting protein behavior in immobilized metal affinity chromatography (IMAC). This article highlights the contribution of solvent-linked parameters in ligand/ligate interactions in IMAC and, hence, brings new clues to selectivity engineering. This leads to the design of a rapid and convenient protocol for the purification of recombinant Actinoplanes missouriensis d-xylose isomerase (XI) and engineered variants produced in Escherichia coli on copper-loaded Chelating Sepharose Fast Flow, an agarose-based matrix derivatized with iminodiacetic acid (IDA) groups. The elucidation of the molecular mechanism by which XI recognizes and interacts with the immobilized metal-chelate complex, IDA-Cu(II), is further made possible by a combination of X-ray structure analysis, molecular modeling, genetic engineering, and mutant characterization. Substitution of lysine for a selected surface histidine residue at position 41 yields a mutant enzyme with near-wild-type properties. The same mutation, however, is shown to completely abolish XI adsorption onto IDA-Cu(II), and hence directly implicates histidine 41 as the predominant protein ligand to the copper-chelate complex. This study not only provides new insights into protocol design for purifying proteins by IMAC, but also describes structural and modeling approaches to analyzing protein surface properties in relation to molecular recognition events.