A novel mechanism of “metal gel-shift” by histidine-rich Ni2+-binding Hpn protein from Helicobacter pylori strain SS1

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) is a universally used method for determining approximate molecular weight (MW) in protein research. Migration of protein that does not correlate with formula MW, termed “gel shifting” appears to be common for histidine-rich proteins but not yet studied in detail. We investigated “gel shifting” in Ni2+-binding histidine-rich Hpn protein cloned from Helicobacter pylori strain SS1. Our data demonstrate two important factors determining “gel shifting” of Hpn, polyacrylamide-gel concentration and metal binding. Higher polyacrylamide-gel concentrations resulted in faster Hpn migration. Irrespective of polyacrylamide-gel concentration, preserved Hpn-Ni2+ complex migrated faster (3–4 kDa) than apo-Hpn, phenomenon termed “metal gel-shift” demonstrating an intimate link between Ni2+ binding and “gel shifting”. To examine this discrepancy, eluted samples from corresponding spots on SDS-gel were analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). The MW of all samples was the same (6945.66±0.34 Da) and identical to formula MW with or without added mass of Ni2+. MALDI-TOF-MS of Ni2+-treated Hpn revealed that monomer bound up to six Ni2+ ions non-cooperatively, and equilibrium between protein-metal species was reliant on Ni2+ availability. This corroborates with gradually increased heterogeneity of apo-Hpn band followed by compact "metal-gel shift" band on SDS-PAGE. In view of presented data metal-binding and “metal-gel shift” models are discussed.

Hpn) were cloned in pET21b. IPTG-induced over-expression of both the proteins was done with or without Ni 2+ added in the culture (Fig S3-A and B). Pellets of 60 µl bacterial cultures were dissolved in 60 µl sample buffer and incubated for 3 min at 100ºC. Final volume of 15 µl loaded in each lane for SDS-PAGE. ELISA experiment was performed using previously described protocol with some modifications (Miura et al. 2008). Different buffers were prepared before starting ELISA experiment (buffer components summarized in Table D).
Flat-bottom 96-well ELISA plates (untreated 96-well microplates from Falcon) were used for coating. Concentration of each protein (Hpn, GFP-Hpn and GFP-His 6 ) was adjusted to 1 µg by dilution with coating buffer to the final volume of 50 µl. Plates were incubated at 4ºC for overnight. Next day, solution was thrown away and 200 µl blocking buffer into each well was added. Then, plate was incubated at 37ºC for 1hr. After incubation, solution was discarded and plate was washed three times by washing buffer. His.Tag® antibody was diluted to standardized concentration (1:500) with dilution buffer [C-terminal specific-anti 6xhistidine monoclonal antibody (9F2) (Wako Japan, product code: 010-21861)] and plate incubated at 37ºC for 1hr after adding 100 μl in each well. After three washes with wash buffer, plate was incubated with horseradish peroxidase-conjugated anti-mouse IgG (GE Healthcare, product code: NA931VS, diluted to 1:1000 in wash buffer) for 1 hr at 37ºC. After similar washing, the substrate ABTS [2,2'-Azinobis(3-ethylbenzothiazoline-6-sulfonic Acid Ammonium Salt) from Wako Japan] dissolved in 0.1 M citrate buffer and hydrogen peroxide (0.03%) was added. Then the plate was incubated for 20 minutes at room temperature.
Reactions were stopped by adding stop buffer (100 µl) in each well. The absorbance at 415 nm was measured using a Spectramax M3 microplate reader (Molecular Devices Co., Sunnyvale, CA). Values obtained (absorbance at 415 nm) for Ni 2+ -treated samples (from average of at least three replications) were normalized against untreated samples and plotted in graph.

Metal-binding to Hpn changes Hpn-antibody interaction
Interaction of His.Tag® antibody with Hpn may not necessarily show similar results for other protein having artificial His.Tag or Hpn conjugated with another protein owing to differential co-ordination geometry of metal-binding and its chemical surrounding. We investigated this possibility using GFP-His 6 and GFP-Hpn (Fig S3-A and B). GFP does not interact with His.Tag® antibody by its own. GFP is comparatively large protein (26.7 kDa) but still positional shift was observed in case of GFP-Hpn expressed in LB medium supplied with Ni 2+ . Western blot data of GFP-His 6 and GFP-Hpn showed almost equal intensity signals in both the cases i.e. with or without Ni 2+ (Fig S3-C).
Recognition site for His.Tag® antibody is His 6 peptide attached at C-terminal of a recombinant protein and it is a linear epitope. Hence, conformational change upon Ni 2+binding to Hpn may lead to altered binding of His.Tag® antibody. This was further examined by ELISA using His.Tag® antibody (Fig S3-D). The SDS-treated and non-treated Hpn protein shown similar results which is compatible with previous observations for several other proteins (Lechtzier et al, 2002;Burgass et al, 2008). The relative detection sensitivity of Ni 2+ -treated protein with His.Tag® antibody in ELISA give order of untagged Hpn < GFP-Hpn < GFP-His 6 . Hence, the variability that we observed in detection of apo-and metalated-Hpn on western blots may have resulted not only from membrane-binding efficiency but also from differential exposure of His-rich region upon metal-binding.
These data signify that the metal-binding to Hpn causes altered binding of His.Tag® antibody, possibly due to change in protein confirmation. The promoter region and hpn gene of strain SS1 was PCR amplified and nucleotide sequence was compared with NCBI data of strain 26695 (GenBank accession number U26361).
Putative promoter elements are shown in a box. The hpn gene region is shown in uppercase letters and mutations at the nucleotide level are shaded in gray. The putative terminator region of transcription is underlined. The promoter region was highly conserved in strain SS1 including Shine-Dalgarno sequence (GGAG) and promoter elements (-10 and -35).