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

Abstract

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-TOFMS). TheMWof 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 proteinmetal 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.