Molecular modeling and dynamics simulation of a histidine-tagged cytochrome b₅

Although an affinity tag such as six consecutive histidines, (His)(6)-tag, has been widely used to obtain high quantity of recombinant proteins, little is known about its influences on heme proteins for lack of structural information. When (His)(6)-tag was introduced to the N-terminus of a small heme protein, cytochrome b(5), experimental results showed the resultant protein, (His)(6)-cyt b(5), has similar property and function to that of isolated cyt b(5). To provide structural information for this observation, we herein performed a structural prediction of (His)(6)-cyt b(5) by molecular modeling in combination with molecular dynamics simulation. The predicted structure, as assessed by a series of criteria with good quality, reveals that the (His)(6)-tag adopts a helical conformation and packs against the hydrophobic core 2 of cyt b(5) through salt bridges, hydrogen bonding and hydrophobic interactions. The heme group, with the axial His ligands slightly rotated, was found to have similar conformation as in isolated cyt b(5), which indicates that the N-terminal (His)(6)-tag does not alter the heme active site, resulting in similar dynamics properties for core 1. This study provides valuable information of interactions between (His)(6)-tag and the rest of the protein, aiding in rational design and application of functional His-tagged proteins.