title: 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-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.
authors.affiliation: Proteo-Science Center, Ehime University, Matsuyama, Japan.
authors.forename: Rahul Mahadev
authors.identifier: http://orcid.org/0000-0003-0691-560X
authors.initials: RM
authors.lastname: Shelake
authors.affiliation: Proteo-Science Center, Ehime University, Matsuyama, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Ito
authors.affiliation: Proteo-Science Center, Ehime University, Matsuyama, Japan.
authors.forename: Junya
authors.initials: J
authors.lastname: Masumoto
authors.affiliation: Laboratory of Molecular Cell Physiology, Faculty of Agriculture, Ehime University, Matsuyama, Japan.|Department of Chemistry, Faculty of Science, Josai University, Saitama, Japan.
authors.forename: Eugene Hayato
authors.initials: EH
authors.lastname: Morita
authors.affiliation: Proteo-Science Center, Ehime University, Matsuyama, Japan.
authors.forename: Hidenori
authors.initials: H
authors.lastname: Hayashi
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D016481:Helicobacter Infections / Q000378:metabolism* / Q000382:microbiology; D016480:Helicobacter pylori / Q000254:growth & development / Q000378:metabolism*; D009532:Nickel / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D001426:Bacterial Proteins; C000597021:Hpn protein, Helicobacter pylori; D009532:Nickel