Aberrantly Large Single-Channel Conductance of Polyhistidine Arm-Containing Protein Nanopores

5 years ago

Aberrantly Large Single-Channel Conductance of Polyhistidine Arm-Containing Protein Nanopores

Motahareh Ghahari Larimi, Avinash Kumar Thakur, Liviu Movileanu, Kristin Gooden

There have been only a few studies reporting on the impact of polyhistidine affinity tags on the structure, function, and dynamics of proteins. Because of the relatively short size of the tags, they are often thought to have little or no effect on the conformation or activity of a protein. Here, using membrane protein design and single-molecule electrophysiology, we determined that the presence of a hexahistidine arm at the N-terminus of a truncated FhuA-based protein nanopore, leaving the C-terminus untagged, produces an unusual increase in the unitary conductance to ∼8 nS in 1 M KCl. To the best of our knowledge, this is the largest single-channel conductance ever recorded with a monomeric β-barrel outer membrane protein. The hexahistidine arm was captured by an anti-polyhistidine tag monoclonal antibody added to the side of the channel-forming protein addition, but not to the opposite side, documenting that this truncated FhuA-based protein nanopore inserts into a planar lipid bilayer with a preferred orientation. This finding is in agreement with the protein insertion in vivo, in which the large loops face the extracellular side of the membrane. The aberrantly large single-channel conductance, likely induced by a greater cross-sectional area of the pore lumen, along with the vectorial insertion into a lipid membrane, will have profound implications for further developments of engineered protein nanopores.

Publisher URL: http://dx.doi.org/10.1021/acs.biochem.7b00577

DOI: 10.1021/acs.biochem.7b00577