[3] Cobalt as probe and label of proteins

Maret, Wolfgang; Vallee, Bertl .

doi:10.1016/0076-6879(93)26005-t

Cited by 160 publications

(32 citation statements)

References 42 publications

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“…The electronic absorption spectrum of Co 2+ -loaded WT CzcP normalized per mole of metal (Figure 4) displays weak transitions in the 450–700 nm region ( ε ≈ 100–200 M −1 cm −1 ), most consistent with tetrahedral, high-spin Co 2+ . 44–46 A strong transition at ~310 nm is also observed and is likely attributable to Cys(thiolate) to Co 2+ ligand-to-metal charge-transfer (LMCT). Literature values for the magnitude of the molar absorptivity of this transition vary, but a similar value to those previously reported 45,47 ( ε ≈ 2000–4000 M −1 cm −1 per Co 2+ ion) is only achieved when normalized against three MBSs, suggesting that all three Co 2+ -loaded sites utilize a Cys(thiolate) in their ligation sphere.…”

Section: Resultsmentioning

confidence: 99%

Metal Selectivity of a Cd-, Co-, and Zn-Transporting P_1B-type ATPase

et al. 2016

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The P1B-ATPases, a family of transmembrane metal transporters important for transition metal homeostasis in all organisms, are subdivided into classes based on sequence conservation and metal specificity. The multifunctional P1B-4-ATPase CzcP is part of the cobalt, zinc, and cadmium resistance system from the metal-tolerant, model organism Cupriavidus metallidurans. Previous work revealed the presence of an unusual soluble metal-binding domain (MBD) at the CzcP N-terminus, but the nature, extent, and selectivity of the transmembrane metal-binding site (MBS) of CzcP have not been resolved. Using homology modeling, we show that four wholly conserved amino acids from the transmembrane (TM) domain (Met254, Ser474, Cys476, and His807) are logical candidates for the TM MBS, which may communicate with the MBD via interactions with the first TM helix. Metal-binding analyses indicate that wild-type (WT) CzcP has three MBSs, and data on N-terminally truncated (ΔMBD) CzcP suggest the presence of a single TM MBS. Electronic absorption and electron paramagnetic resonance spectroscopic analyses of ΔMBD CzcP and variant proteins thereof provide insight into the details of Co2+ coordination by the TM MBS. These spectroscopic data, combined with in vitro functional studies of WT and variant CzcP proteins, show that the side chains of Met254, Cys476, and His807 contribute to Cd2+, Co2+, and Zn2+ binding and transport, whereas the side chain of Ser474 appears to play a minimal role. By comparison to other P1B-4-ATPases, we suggest that an evolutionarily adapted flexibility in the TM region likely afforded CzcP the ability to transport Cd2+ and Zn2+ in addition to Co2+.

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Section: Resultsmentioning

confidence: 99%

Metal Selectivity of a Cd-, Co-, and Zn-Transporting P_1B-type ATPase

et al. 2016

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“…1 The cobalt ion typically adopts a high-spin ( hs ) configuration and a coordination geometry similar to that of the native zinc complex. In contrast to Zn(II), which is only accessible by x-ray absorption spectroscopy (XAS), 2-9 complexes of hs Co(II), including those of enzymes, are amenable to a wide array of spectroscopies, including optical spectroscopy, XAS, 10-17 magnetic circular dichroism (MCD), 18-24 , EPR, 15,16,21,25-44 NMR 43-68 and, more recently, electron-nuclear double resonance (ENDOR) 69 and high-frequency/field EPR (HF/F-EPR).…”

Section: Introductionmentioning

confidence: 99%

Integrated Paramagnetic Resonance of High-Spin Co(II) in Axial Symmetry: Chemical Separation of Dipolar and Contact Electron−Nuclear Couplings

2008

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Integrated paramagnetic resonance, utilizing EPR, NMR and ENDOR, of a series of cobalt bis-trispyrazolylborates, Co(Tpx)2, are reported. Systematic substitutions at the ring carbons and on the apical boron provide a unique opportunity to separate through-bond and through-space contributions to the NMR hyperfine shifts for the parent, unsubstituted Tp complex. A simple relationship between the chemical shift difference (δH − δMe) and the contact shift of the proton in that position is developed. This approach allows independent extraction of the isotropic hyperfine coupling, Aiso, for each proton in the molecule. The Co··H contact coupling energies derived from the NMR, together with the known metrics of the compounds, were used to predict the ENDOR couplings at gζ. Proton ENDOR data is presented that shows good agreement with the NMR-derived model. ENDOR signals from all other magnetic nuclei in the complex (14N, coordinating and non-coordinating, 11B and 13C) are also reported.

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“…For instance, the aqua complex of Co 2+ has an exchange rate of 3×10 6 s −1 while the Co 3+ complex has an exchange rate of less than 10 −6 s −1 (Scheme 1). 27 The idea to use Co 3+ as a mediator ion between NTA and the His6-tag has been inspired by studies on metalloenzymes where the substitution of the native metal ion by exchange-inert metal ions such as Co 3+ and Cr 3+ has been particularly useful in elucidating enzymatic mechanisms, differentiating between multiple metal sites in an enzyme, and providing insight into the local environment of the metal binding pocket and overall structure 28, 29. In addition, complexes of bioactive ligands with exchange-inert metal ions have been used as prodrugs that are only activated after entering the cell 30, 31.…”

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confidence: 99%

Cobalt(III) as a Stable and Inert Mediator Ion between NTA and His6‐Tagged Proteins

Wegner

Spatz

2013

Angew Chem Int Ed

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Don't let go! The Co3+‐mediated interaction between nitrilotriacetic acid (NTA) and the His6‐tag is so stable and inert towards ligand exchange that it has a half‐life of 7 days in the presence of imidazole and survives even under strongly chelating as well as reducing conditions, unlike the commonly used Ni2+ or Co2+ complexes. Possible applications include the separation of labeled proteins and the stable immobilization of proteins on surfaces.

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[3] Cobalt as probe and label of proteins

Cited by 160 publications

References 42 publications

Metal Selectivity of a Cd-, Co-, and Zn-Transporting P_1B-type ATPase

Metal Selectivity of a Cd-, Co-, and Zn-Transporting P_1B-type ATPase

Integrated Paramagnetic Resonance of High-Spin Co(II) in Axial Symmetry: Chemical Separation of Dipolar and Contact Electron−Nuclear Couplings

Cobalt(III) as a Stable and Inert Mediator Ion between NTA and His6‐Tagged Proteins

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[3] Cobalt as probe and label of proteins

Cited by 160 publications

References 42 publications

Metal Selectivity of a Cd-, Co-, and Zn-Transporting P1B-type ATPase

Metal Selectivity of a Cd-, Co-, and Zn-Transporting P1B-type ATPase

Integrated Paramagnetic Resonance of High-Spin Co(II) in Axial Symmetry: Chemical Separation of Dipolar and Contact Electron−Nuclear Couplings

Cobalt(III) as a Stable and Inert Mediator Ion between NTA and His6‐Tagged Proteins

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Product

Resources

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Metal Selectivity of a Cd-, Co-, and Zn-Transporting P_1B-type ATPase

Metal Selectivity of a Cd-, Co-, and Zn-Transporting P_1B-type ATPase