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

Abstract

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 their relatively short size, they are often assumed to have little or no effect on the conformation and 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 up to ~8 nS in 1 M KCl. To 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.

Real-time measurement of protein–protein interactions at single-molecule resolution using a biological nanopore

“…All the designed genes were constructed using conventional and assembly PCR techniques, and cloned into the expression vector pPR-IBA1 using respective restriction sites. bn(ggs) 2 t-fhua , which encoded barnase (Bn) 34 at the N-terminus of the heavily truncated t-FhuA protein 20 via a flexible Gly/Ser-rich tether and BsaI site, was prepared using the bn and t-fhua genes, as well as three PCR reactions. The first PCR reaction was performed using bn as a template DNA, the forward primer (1) and reverse primer (2).…”

“…Because the detection of binding events occurs outside the ‘voltage-drop region’ of the nanopore, we can apply our sensor to detect transient PPIs in mammalian serum. A single-polypeptide chain protein was created using t-FhuA 20 , a monomeric β-barrel scaffold that is a truncated version of ferric hydroxamate uptake component A (FhuA) 21 from Escherichia coli (Fig. 1a1).…”

“…An H102A mutant of Bn was used, because this lacks ribonuclease activity 22, 23 which in turn ensures that Bn-FhuA fusion proteins are not toxic to the expression host (ONLINE METHODS). The 455-residue t-FhuA scaffold formed a transmembrane pore, 20 facilitating a quiet single-channel electrical signature for long periods when added to the cis side of the chamber (Supplementary Fig. 1).…”

“…26, 27 The pore is inserted such that the loops face the trans side and the β turns face the cis side (Fig. 1a1) 20 .…”

High‐Throughput Screening of Protein‐Detergent Complexes Using Fluorescence Polarization Spectroscopy

“…In these examples, the proteins were polyhistidine‐tagged and purified using affinity chromatography. Alternatively, β barrels can be purified by tag‐free anion‐exchange chromatography (Thakur, Larimi, Gooden, & Movileanu, ; Thakur & Movileanu, ; Wolfe, Mohammad, Thakur, & Movileanu, ). We think that the polyhistidine tag does not significantly affect the detergent solubilization properties of a β barrel protein.…”