The Effects of Histidine Tags on the Energy Landscape on Acyl Co‐A Binding Protein

Abstract

Protein epitope tags play an important role in protein characterization, allowing for easy detection, tracking and isolation in cellular and biochemical experiments. A polyhistidine tag is an amino acid motif that consists of at least six histidine (His) residues, often located at the N‐ or C‐terminus of the protein. Affinity tags, like His tags, are routinely employed as convenient means of purifying recombinantly expressed proteins. However there have been increasing reports suggesting that His tags interfere with the structure and function of proteins. We hypothesize that His tags affect the energy landscape of proteins. Acyl Co‐A Binding Protein (ACBP) is a single‐domain protein that is thought to fold in a two‐state process To test our hypothesis we studied the stability and folding kinetics of ACBP with differentially located His tags in the presence of buffers with varying pHs with a fluorimeter and a stopped‐flow spectrometer. First, we found that buffer pH differentially affected the charge of the His tag: At a pH of 5.3 Histidine is protonated and at 7 Histidine is deprotonated. Second, we observed a significant difference in the kinetics and thermodynamics of the C‐Terminus His tagged ACBP relative to the taglesss ACBP. In a pH of 5.3, the C‐Term His tagged ACBP was less stable whereas in a pH of 7 it was more stable. On the other hand, there was no significant difference in the thermodynamics or the kinetics of the N‐term ACBP to the tagless ACBP. Taken together our studies suggest that the location of the His tag (and ionic environment) markedly affected the energy landscape of ACBP. Support or Funding Information DePauw University‐PDF