title: Correlation between protein binding strength on immobilized metal affinity chromatography and the histidine-related protein surface structure.
abstract: Immobilized metal affinity chromatography (IMAC) was investigated for its ability to characterize the histidine-related surface structure of a protein, that is, a histidine residue's surface accessibility and its potential involvement in intramolecular interactions. T4 lysozyme was chosen as the model protein. Seven amino acid sites were selected on the basis of their relative surface accessibility, and they were substituted with histidine via site-directed protein mutagenesis to generate seven T4 lysozyme variants, each containing only one histidine residue on its surface, with various surface accessibility. IMAC was then used to experimentally quantify the interaction of each lysozyme variant with immobilized copper ions. A direct correlation was shown between the protein binding affinity and the surface accessibility of the histidine residue. Of all the lysozyme variants, K83H and K147H showed unusually low binding strength, as compared with variants having a histidine residue with a similar surface accessibility. However, with the aid of molecular modeling, their relatively low binding affinities were predicted to be the result of the involvement of the histidine residue in intramolecular interactions. In contrast to previously reported results, our results showed that lysozyme still binds to the IMAC column, even if its histidine residue is involved in intramolecular bonding, such as a hydrogen bond, albeit at reduced strength, as compared with the variant containing a histidine residue with a similar surface accessibility.
authors.affiliation: Department of Biological Systems Engineering, and Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA.
authors.forename: Lakshmi P
authors.initials: LP
authors.lastname: Pathange
authors.forename: David R
authors.initials: DR
authors.lastname: Bevan
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Larson
authors.forename: Chenming
authors.initials: C
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D006639:Histidine / Q000378:metabolism*; D008670:Metals; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D008670:Metals; D011506:Proteins; D006639:Histidine