1 research outputs found
Aberrantly Large Single-Channel Conductance of Polyhistidine Arm-Containing Protein Nanopores
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 <i>in vivo</i>, 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