List of histidine-rich Ni²⁺-binding proteins showing gel shifting anomaly.

<p>List of histidine-rich Ni²⁺-binding proteins showing gel shifting anomaly.</p

Effect of polyacrylamide-gel concentration on migration speed of recombinant Hpn without or with metal-treatment on SDS-PAGE.

<p>A. Relative position of apo- and Ni²⁺-treated Hpn protein depending on the polyacrylamide percentage (15, 18, 20 and 22.5%) in gels. Lane M is marker proteins standard from Nacalai Tesque (200, 116, 66, 45, 31, 21.5, 14.4, 6.5 kDa from top to bottom respectively). The SDS-gel electrophoresis was done till bromophenol blue dye reached to the bottom in all gels. Therefore, theoretical MW values for marker proteins- lysozyme (14.4 kDa) and Trypsin inhibitor (21.5 kDa) were taken into consideration for estimation of apparent MW using ImageJ software (<b>protocol provided in Supplementary information as Annexure A</b>). Red dotted line depicts expected position corresponding to theoretical MW (~6.9 kDa). B. The apparent MW of apo- and Ni²⁺-treated Hpn separated on different polyacrylamide-gel concentrations was estimated by comparing relative migration distance of Hpn with globular marker proteins on SDS-PAGE.</p

Ni²⁺ tolerance, cell survival, and accumulation in Hpn-expressing <i>E</i>. <i>coli</i>.

<p>Effect of Ni²⁺ on growth of <i>E</i>. <i>coli</i> in Luria-Bertani (LB) (panel A) and M9 medium (panel D). Growth curve was plotted in terms of optical density (OD) against amount of Ni²⁺added to culture. Cell survival under Ni²⁺ stress analyzed by dots blot as shown in panel B (LB) and E (M9). Panel C (LB) and F (M9) represent the intracellular Ni²⁺ content in <i>E</i>. <i>coli</i> expressing <i>hpn</i> gene compare to that of without <i>hpn</i> gene.</p

MALDI-TOF-MS analysis of Ni²⁺ binding to Hpn.

<p>A. Spectra obtained with increasing concentrations of Ni²⁺ (1:0, 1:1, 1:2.5, 1:5, 1:10, 1:20 and 1:40, mol equivalent from top to bottom, respectively) added to apo-Hpn (25 μM) shown in the right panel, and enlarged view of three representative spectra (1:0, 1:10 and 1:40) shown with molecular mass of each peak in the left side. Numbers above dotted line correspond to the number of Ni²⁺ bound to Hpn protein. B. Mass difference calculated between adjacent peaks was found to be approximately equal to the molecular mass of the Ni²⁺ ion (58.69) with the loss of two H⁺ atoms (molecular weight [MW] of H⁺ = 1.00794) upon metal binding. C. Model of Ni²⁺ ion binding to Hpn. Order of occurrence has drawn on the basis of peak intensity obtained in MALDI-TOF-MS data.</p

Amino acid sequence, overexpression and purification of recombinant Hpn.

<p>Lane M, LMW protein marker standards (GE Healthcare; MW from top to bottom: 97, 66, 45, 30, 20.1 and 14.4 kDa); black arrows depicting apo-Hpn and white arrows showing probable Ni²⁺-bound Hpn protein in all panels. A. Amino acid sequence of Hpn. Histidine residues are highlighted in bold. Stretches of six and seven histidines are highlighted in green and pentapetide repeats (EEGCC) are underlined. B. SDS-PAGE of Hpn expression with or without Ni²⁺ added in the culture (polyacrylamide-gel 20%). Pellets of 60 μl bacterial cultures were dissolved in 60 μl of 1X Laemmli buffer and boiled for 3 min at 100°C. Final volume of 15 μl loaded in each lane. C, D and E. Elution profile of purified Hpn checked by loading protein fractions on SDS-PAGE (polyacrylamide-gel 15%). Lanes 1 to 10, fractions of purified protein eluted with 400 mM imidazole (C). Elution profiles of desalted fractions of Hpn without EDTA treatment (D) and with EDTA-treatment (E) were analyzed. Equal volume of 2X Laemmli buffer was added to each eluted fraction and then boiled for 3 min at 100°C. Total 10 μl applied in each lane in C, D and E.</p

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

<div><p>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 <i>Helicobacter pylori</i> 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.</p></div

Confirmation of “Metal gel-shift” mechanism.

<p>A. Effect of EDTA and Ni²⁺ ion treatment on migration rate of recombinant Hpn in SDS-PAGE (polyacrylamide-gel 20%). Lane M, protein marker (GE Healthcare; MW from top to bottom: 97, 66, 45, 30, 20.1 and 14.4 kDa); lane 1 and 2, Hpn before and after boiling (3 min at 100°C), respectively; lanes 3 and 4, EDTA-treated Hpn without and with boiling, respectively; lanes 5 and 6, Ni²⁺-treated Hpn without and with boiling, respectively. B. The SDS-PAGE analysis of partially-metalated-Hpn (25 μM) treated with increasing concentration of Ni²⁺ (1:0, 1:0.8, 1:1.2, 1:1.6, 1:2.0, 1:2.4, 1:2.8, 1:3.2, 1:3.6, 1:4.0, 1:6.0 and 1:8.0). Lane M is marker proteins standard from Nacalai Tesque (200, 116, 66, 45, 31, 21.5, 14.4, 6.5 kDa from top to bottom respectively). Equal volume of heat-denatured protein applied in each lane. C. Scheme used for MALDI-TOF-MS analysis of Hpn protein that was heat denatured in Laemmli buffer. MS data was measured for Hpn treated with or without Ni²⁺ ion (1:6 mol equivalent ratios). Further, MS data for Hpn (with or without Ni²⁺) treated in Laemmli buffer (before and after SDS-PAGE) was measured. Even though some interference due to adducts was observed in samples treated with Laemmli buffer or gel-eluted fractions, metalated peaks (showing Hpn-Ni²⁺ complexes) were distinct. The occurrence of metalated peaks was observed only for Ni²⁺-treated Hpn in all the conditions.</p

Western blot and molecular mass analysis of Hpn with MALDI-TOF-MS.

<p>A. Western blot of Hpn was done using His.Tag^® monoclonal antibody. In left panel, X-ray sheet showing ECL detection result of recombinant Hpn (with and without Ni²⁺) and right panel showing same CBB-stained-PVDF membrane used for ECL detection. B. Sharp peak of monomeric Hpn together with neighboring small peak of matrix adduct, and three oligomeric species with lower intensity was observed in Hpn spectrum. The monomeric and three oligomeric species: M⁺, 2M⁺, 3M⁺, and 4M⁺ with masses of <i>m/z</i> 6942.09, 13874.39, 20807.59, 27740.62 respectively are shown in inset. C. Molecular mass of Hpn was measured (<i>m/z</i> 6946.05) using two different internal standards (insulin and apomyoglobin), which showed peaks for protonated and doubly charged species. Average MW of recombinant Hpn (without methionine) determined (6945.66±0.34) is showing almost negligible difference (0.35) compared to theoretical MW (6946.01).</p

Probable interrelationship between differential electrophoretic mobility of Hpn and Ni²⁺ binding.

<p>Hpn may not have a definite form in the absence of Ni²⁺ (A). After denaturation (B), smaller amounts of SDS binding/stacking behavior/larger hydrodynamic radius as well as a combination of some or all of these conditions (C) might have resulted in slower migration on SDS-PAGE (scheme highlighted with yellow background). Ni²⁺-treated Hpn forms a more compact structure (D). Pictorial structure of metalated Hpn is drawn to explain the model. MALDI spectra showed a partial Ni²⁺ bound form (E) in denatured SDS-PAGE. Altered binding of SDS (F) caused by replacement of protein-protein to protein-SDS contacts (inhibiting stacking behavior) and/or degree of compactness (or reduced hydrodynamic radius) may be key factors responsible for “metal gel-shift” (scheme highlighted with green background). β-ME, β-mercaptoethanol; EDTA, ethylene diaminetetraacetic acid.</p

Spirobis(pentagerma[1.1.1]propellane): A Stable Tetraradicaloid

In this contribution, we report a spirobis­(pentagerma[1.1.1]­propellane) derivative as a novel type of molecular architecture in cluster chemistry that features two spiro-fused [1.1.1]­propellane units and represents a stable tetraradicaloid species. The crucial issue of the nature of the interaction between the germanium bridgeheads was probed computationally, revealing weak bonding interactions between the formally unpaired electrons