scholarly journals Mapping of a copper-binding site on the small CP12 chloroplastic protein of Chlamydomonas reinhardtii using top-down mass spectrometry and site-directed mutagenesis

2009 ◽  
Vol 419 (1) ◽  
pp. 75-86 ◽  
Author(s):  
Jenny Erales ◽  
Brigitte Gontero ◽  
Julian Whitelegge ◽  
Frédéric Halgand
Keyword(s):  
Metal Ion ◽  
Calvin Cycle ◽  
Computational Modelling ◽  
Copper Ion ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Copper Binding ◽  
Metal Ion Binding ◽  
Top Down ◽  
Copper Ligands

CP12 is a small chloroplastic protein involved in the Calvin cycle that was shown to bind copper, a metal ion that is involved in the transition of CP12 from a reduced to an oxidized state. In order to describe CP12's copper-binding properties, copper-IMAC experiments and site-directed mutagenesis based on computational modelling, were coupled with top-down MS [electrospray-ionization MS and MS/MS (tandem MS)]. Immobilized-copper-ion-affinity-chromatographic experiments allowed the primary characterization of the effects of mutation on copper binding. Top-down MS/MS experiments carried out under non-denaturing conditions on wild-type and mutant CP12–Cu2+ complexes then allowed fragment ions specifically binding the copper ion to be determined. Comparison of MS/MS datasets defined three regions involved in metal ion binding: residues Asp16–Asp23, Asp38–Lys50 and Asp70–Glu76, with the two first regions containing selected residues for mutation. These data confirmed that copper ligands involved glutamic acid and aspartic residues, a situation that contrasts with that obtaining for typical protein copper chelators. We propose that copper might play a role in the regulation of the biological activity of CP12.

scholarly journals Site-Directed Mutagenesis Identifies a Molecular Switch Involved in Copper Sensing by the Histidine Kinase CinS in Pseudomonas putida KT2440

2009 ◽  
Vol 191 (16) ◽  
pp. 5304-5311 ◽  
Author(s):  
Davide Quaranta ◽  
Megan M. McEvoy ◽  
Christopher Rensing
Keyword(s):  
Pseudomonas Putida ◽  
Histidine Kinase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Copper Binding ◽  
Wild Type ◽  
Two Component System ◽  
Pseudomonas Putida Kt2440 ◽  
Component System ◽  
Two Component

ABSTRACT In the presence of copper, Pseudomonas putida activates transcription of cinAQ via the two-component system CinS-CinR. The CinS-CinR TCS was responsive to 0.5 μM copper and was specifically activated only by copper and silver. Modeling studies of CinS identified a potential copper binding site containing H37 and H147. CinS mutants with H37R and H147R mutations had an almost 10-fold reduced copper-dependent induction of cinAQ compared to the wild type.

Identification of the ferroxidase centre of Escherichia coli bacterioferritin

1995 ◽  
Vol 312 (2) ◽  
pp. 385-392 ◽  
Author(s):  
N E Le Brun ◽  
S C Andrews ◽  
J R Guest ◽  
P M Harrison ◽  
G R Moore ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metal Ion ◽  
Oxidation Mechanism ◽  
Site Directed Mutagenesis ◽  
Ion Binding ◽  
Metal Ion Binding ◽  
Dinuclear Iron ◽  
Ferroxidase Activity ◽  
A Site ◽  
R2 Subunit

The bacterioferritin (BFR) of Escherichia coli takes up iron in the ferrous form and stores it within its central cavity as a hydrated ferric oxide mineral. The mechanism by which oxidation of iron (II) occurs in BFR is largely unknown, but previous studies indicated that there is ferroxidase activity associated with a site capable of forming a dinuclear-iron centre within each subunit [Le Brun, Wilson, Andrews, Harrison, Guest, Thomson and Moore (1993) FEBS Lett. 333, 197-202]. We now report site-directed mutagenesis experiments based on a putative dinuclear-metal-ion-binding site located within the BFR subunit. The data reveal that this dinuclear-iron centre is located at a site within the four-alpha-helical bundle of each subunit of BFR, thus identified as the ferroxidase centre of BFR. The metal-bound form of the centre bears a remarkable similarity to the dinuclear-iron sites of the hydroxylase subunit of methane mono-oxygenase and the R2 subunit of ribonucleotide reductase. Details of how the dinuclear centre of BFR is involved in the oxidation mechanism were investigated by studying the inhibition of iron (II) oxidation by zinc (II) ions. Data indicate that zinc (II) ions bind at the ferroxidase centre of apo-BFR in preference to iron (II), resulting in a dramatic reduction in the rate of oxidation. The mechanism of iron (II) oxidation is discussed in the light of this and previous work.

scholarly journals Metal Ion Binding to the Amyloid β Monomer Studied by Native Top-Down FTICR Mass Spectrometry

2019 ◽  
Vol 30 (10) ◽  
pp. 2123-2134 ◽  
Author(s):  
Frederik Lermyte ◽  
James Everett ◽  
Yuko P. Y. Lam ◽  
Christopher A. Wootton ◽  
Jake Brooks ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Metal Ion ◽  
Amyloid Β ◽  
Ion Binding ◽  
Metal Ion Binding ◽  
Top Down ◽  
Fticr Mass Spectrometry

scholarly journals Identification and characterization of the conserved nucleoside-binding sites in the Epstein-Barr virus thymidine kinase

2004 ◽  
Vol 379 (3) ◽  
pp. 795-803 ◽  
Author(s):  
Chung-Chun WU ◽  
Min-Che CHEN ◽  
Ya-Ru CHANG ◽  
Tsuey-Ying HSU ◽  
Jen-Yang CHEN
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Thymidine Kinase ◽  
Crucial Role ◽  
Metal Ion ◽  
Epstein Barr Virus ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Barr Virus ◽  
Epstein Barr

Thymidine kinase (TK), encoded by EBV (Epstein–Barr virus), is an attractive target for antiviral therapy and provides a novel approach to the treatment of EBV-associated malignancies. Despite the extensive use of nucleoside analogues for the treatment of viral infections and cancer, the structure–function relationship of EBV TK has been addressed rarely. In the absence of any structural information, we sought to identify and elucidate the functional roles of amino acids in the nucleoside-binding site using site-directed mutagenesis. Through alignment with other human herpesviral TK protein sequences, we predicted that certain conserved regions comprise the nucleoside-binding site of EBV TK and, through site-directed mutagenesis, showed significant changes in activity and binding affinity for thymidine of site 3 (-DRH-) and 4 (-VFP-) mutants. For site 3, only mutants D392E (Asp392→Glu) and R393H retain activity, indicating that a negative charge is important for Asp392 and a positive charge is required for Arg393. The increased binding affinities of these two mutants for 3´-deoxy-2´,3´-didehydrothymidine suggest that the two residues are also important for substrate selection. Interestingly, the changed metal-ion usage pattern of D392E reveals that Asp392 plays multiple roles in this region. His394 cannot be compensated by other amino acids, also indicating a crucial role. In site 4, the F402Y mutant retains full activity; however, F402S retains only 60% relative activity. Strikingly, when Phe402 is substituted with serine residue, the original preferred pyrimidine substrates, such as 3´-azido-3´-deoxythymidine, iododeoxyuridine and β-l-5-iododioxolane uracil (l-form substrate), have decreased competitiveness with thymidine, suggesting that Phe402 plays a crucial role in substrate specificity and that the aromatic ring is important for function.

scholarly journals The Advantages of EPR Spectroscopy in Exploring Diamagnetic Metal Ion Binding and Transfer Mechanisms in Biological Systems

2021 ◽  
Vol 8 (1) ◽  
pp. 3
Author(s):  
Shelly Meron ◽  
Yulia Shenberger ◽  
Sharon Ruthstein
Keyword(s):  
Ligand Binding ◽  
Conformational Changes ◽  
Epr Spectroscopy ◽  
Metal Ion ◽  
Copper Ion ◽  
Biological Processes ◽  
Ion Transfer ◽  
Metal Ion Binding ◽  
Cellular Mechanisms ◽  
Paramagnetic Resonance

Electron paramagnetic resonance (EPR) spectroscopy has emerged as an ideal biophysical tool to study complex biological processes. EPR spectroscopy can follow minor conformational changes in various proteins as a function of ligand or protein binding or interactions with high resolution and sensitivity. Resolving cellular mechanisms, involving small ligand binding or metal ion transfer, is not trivial and cannot be studied using conventional biophysical tools. In recent years, our group has been using EPR spectroscopy to study the mechanism underlying copper ion transfer in eukaryotic and prokaryotic systems. This mini-review focuses on our achievements following copper metal coordination in the diamagnetic oxidation state, Cu(I), between biomolecules. We discuss the conformational changes induced in proteins upon Cu(I) binding, as well as the conformational changes induced in two proteins involved in Cu(I) transfer. We also consider how EPR spectroscopy, together with other biophysical and computational tools, can identify the Cu(I)-binding sites. This work describes the advantages of EPR spectroscopy for studying biological processes that involve small ligand binding and transfer between intracellular proteins.

scholarly journals Helicobacter pylori UreE, a urease accessory protein: specific Ni2+- and Zn2+-binding properties and interaction with its cognate UreG

2009 ◽  
Vol 422 (1) ◽  
pp. 91-100 ◽  
Author(s):  
Matteo Bellucci ◽  
Barbara Zambelli ◽  
Francesco Musiani ◽  
Paola Turano ◽  
Stefano Ciurli
Keyword(s):  
Helicobacter Pylori ◽  
Light Scattering ◽  
Metal Ion ◽  
Site Directed Mutagenesis ◽  
Size Exclusion ◽  
Titration Calorimetry ◽  
Directed Mutagenesis ◽  
Accessory Protein ◽  
Oligomeric State ◽  
Binding Properties

The persistence of Helicobacter pylori in the hostile environment of the human stomach is ensured by the activity of urease. The essentiality of Ni2+ for this enzyme demands proper intracellular trafficking of this metal ion. The metallo-chaperone UreE promotes Ni2+ insertion into the apo-enzyme in the last step of urease maturation while facilitating concomitant GTP hydrolysis. The present study focuses on the metal-binding properties of HpUreE (Helicobacter pylori UreE) and its interaction with the related accessory protein HpUreG, a GTPase involved in the assembly of the urease active site. ITC (isothermal titration calorimetry) showed that HpUreE binds one equivalent of Ni2+ (Kd=0.15 μM) or Zn2+ (Kd=0.49 μM) per dimer, without modification of the protein oligomeric state, as indicated by light scattering. Different ligand environments for Zn2+ and Ni2+, which involve crucial histidine residues, were revealed by site-directed mutagenesis, suggesting a mechanism for discriminating metal-ion-specific binding. The formation of a HpUreE–HpUreG protein complex was revealed by NMR spectroscopy, and the thermodynamics of this interaction were established using ITC. A role for Zn2+, and not for Ni2+, in the stabilization of this complex was demonstrated using size-exclusion chromatography, light scattering, and ITC experiments. A calculated viable structure for the complex suggested the presence of a novel binding site for Zn2+, actually detected using ITC and site-directed mutagenesis. The results are discussed in relation to available evidence of a UreE–UreG functional interaction in vivo. A possible role for Zn2+ in the Ni2+-dependent urease system is envisaged.

Copper-Induced Oligomerization of Peptides: A Model Study

2007 ◽  
Vol 13 (5) ◽  
pp. 331-337 ◽  
Author(s):  
Gitta Schlosser ◽  
Raluca Stefanescu ◽  
Michael Przybylski ◽  
Manuela Murariu ◽  
Ferenc Hudecz ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Metal Ion ◽  
Ion Binding ◽  
Ionization Mass ◽  
Copper Binding ◽  
Base Peak ◽  
Metal Ion Binding ◽  
Model Study ◽  
Alkaline Conditions ◽  
Singly Charged

In this work, copper-binding of the tetraglycine peptide (Gly–Gly–Gly–Gly) was studied by electrospray ionization mass spectrometry. Experiments were performed under alkaline conditions, in the presence of ethanolamine (pH 10.95). We observed that the presence of copper(II) ions induces the aggregation of the peptide and the formation of copper-bound complexes with higher molecular mass is favored, such as the oligomer complexes [3M + 2Cu – 3H]+ and [4M + 3Cu – 5H]+. At 1:1 peptide–copper(II) ion ratio, the singly charged [3M + 2Cu – 3H]+ oligomer complex is the base peak in the mass spectrum. Metal ion-induced oligomerization of neurotoxic peptides is well known in the literature; however, there are very few examples in which such oligomerization was directly observed by mass spectrometry. Our results show that application of short peptides can be useful to study the mechanism of metal ion binding and metal ion-induced oligomerization of peptides.

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