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-r...

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Main Authors: Rahul Mahadev Shelake, Yuki Ito, Junya Masumoto, Eugene Hayato Morita, Hidenori Hayashi
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2017-01-01
Series:PLoS ONE
Online Access:http://europepmc.org/articles/PMC5312948?pdf=render
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spelling doaj-f02ba47722ef47148e2a616aa13217fc2020-11-25T00:01:49ZengPublic Library of Science (PLoS)PLoS ONE1932-62032017-01-01122e017218210.1371/journal.pone.0172182A novel mechanism of "metal gel-shift" by histidine-rich Ni2+-binding Hpn protein from Helicobacter pylori strain SS1.Rahul Mahadev ShelakeYuki ItoJunya MasumotoEugene Hayato MoritaHidenori HayashiSodium 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.http://europepmc.org/articles/PMC5312948?pdf=render
collection DOAJ
language English
format Article
sources DOAJ
author Rahul Mahadev Shelake
Yuki Ito
Junya Masumoto
Eugene Hayato Morita
Hidenori Hayashi
spellingShingle Rahul Mahadev Shelake
Yuki Ito
Junya Masumoto
Eugene Hayato Morita
Hidenori Hayashi
A novel mechanism of "metal gel-shift" by histidine-rich Ni2+-binding Hpn protein from Helicobacter pylori strain SS1.
PLoS ONE
author_facet Rahul Mahadev Shelake
Yuki Ito
Junya Masumoto
Eugene Hayato Morita
Hidenori Hayashi
author_sort Rahul Mahadev Shelake
title A novel mechanism of "metal gel-shift" by histidine-rich Ni2+-binding Hpn protein from Helicobacter pylori strain SS1.
title_short A novel mechanism of "metal gel-shift" by histidine-rich Ni2+-binding Hpn protein from Helicobacter pylori strain SS1.
title_full A novel mechanism of "metal gel-shift" by histidine-rich Ni2+-binding Hpn protein from Helicobacter pylori strain SS1.
title_fullStr A novel mechanism of "metal gel-shift" by histidine-rich Ni2+-binding Hpn protein from Helicobacter pylori strain SS1.
title_full_unstemmed A novel mechanism of "metal gel-shift" by histidine-rich Ni2+-binding Hpn protein from Helicobacter pylori strain SS1.
title_sort novel mechanism of "metal gel-shift" by histidine-rich ni2+-binding hpn protein from helicobacter pylori strain ss1.
publisher Public Library of Science (PLoS)
series PLoS ONE
issn 1932-6203
publishDate 2017-01-01
description 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.
url http://europepmc.org/articles/PMC5312948?pdf=render
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