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1.
Molecular dynamics simulations of two tandem octarepeats from the mammalian prion protein: fully Cu2+-bound and metal-free forms
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Molecular dynamics simulations have been conducted on a model fragment (Ac-PHGGGWGQPHGGGW-NH2) of the prion protein octarepeat domain, both in the Cu2+-bound and metal-free forms. The copper-bound models are based on the consensus structure of the core Cu2+-binding site of an individual octarepeat, relevant to the fully Cu2+-occupied prion protein octarepeat region. The model peptides contain Cu2+ bound through a His imidazole ring and two deprotonated amide N-atoms in the peptide backbone supplied by the following two Gly residues. Both the copper-bound and metal-free models have been simulated with the OPLS all-atom force field with the GROMACS molecular dynamics package. These simulations, with two tandem copper-binding sites, represent the minimum model necessary to observe potential structuring between the copper-binding sites in the octarepeat region. The GWGQ residues constitute a flexible linker region that predominantly adopts a turn, serving to bring adjacent His residues into close proximity. The consequent formation of stable structures demonstrates that the copper-bound octarepeat region allows the copper-coordinating sites to come into van der Waals contact, packing into particular orientations to further stabilize the bend in the GWGQ linker region. 相似文献
2.
Sara Furlan Giovanni La Penna Francesco Guerrieri Silvia Morante Gian Carlo Rossi 《Journal of biological inorganic chemistry》2007,12(4):571-583
The human prion protein binds Cu2+ ions in the octarepeat domain of the N-terminal tail up to full occupancy at pH 7.4. Recent experiments have shown that the
HGGG octarepeat subdomain is responsible for holding the metal bound in a square-planar configuration. By using first principle
ab initio molecular dynamics simulations of the Car–Parrinello type, the coordination of copper to the binding sites of the
prion protein octarepeat region is investigated. Simulations are carried out for a number of structured binding sites. Results
for the complexes Cu(HGGGW)(wat), Cu(HGGG), and [Cu(HGGG)]2 are presented. While the presence of a Trp residue and a water molecule does not seem to affect the nature of the copper
coordination, high stability of the bond between copper and the amide nitrogen of deprotonated Gly residues is confirmed in
all cases. For the more interesting [Cu(HGGG)]2 complex, a dynamically entangled arrangement of the two domains with exchange of amide nitrogen bonds between the two copper
centers emerges, which is consistent with the short Cu–Cu distance observed in experiments at full copper occupancy. 相似文献
3.
《Journal of inorganic biochemistry》1986,26(2):117-125
Myoglobin of Aplysia brasiliana (MbApB) has been recently purified and characterized and it was shown that the amino acid content is quite different from other myoglobins. A large number of aromatic residues was observed together with the existence of a unique histidine at the proximal heme position. Because of the numerous differences in the amino acid sequence between MbApB and whale myoglobin, it was interesting to investigate the interaction of metal ions like Cu2+ and Mn2+ with MbApB. In the present work Cu2+ complexes with Met-MbApB were studied and show a pH transition between different forms of coordination as revealed by EPR measurements. At high pH the EPR spectrum shows the coordination of the metal to at least four nitrogens from ϵ-NH3 lysine residues. At lower pH in the range 6.0–9.0 the copper binding site shows a pK change of some of the residues involved in metal coordination. Addition of one equivalent Cu2+ per protein does not alter the iron EPR signal. The manganese ion has one binding site in MbApB and a binding constant Ka = ( 11.5 ± 0.8) 103M−1. The binding of Cu2+ to MbApB is stronger than Mn2+, KaCu2+ >KaMn2+. 相似文献
4.
Daniel Mattle Limei Zhang Oleg Sitsel Lotte Thue Pedersen Maria Rosa Moncelli Francesco Tadini-Buoninsegni Pontus Gourdon Douglas C Rees Poul Nissen Gabriele Meloni 《EMBO reports》2015,16(6):728-740
Cells regulate copper levels tightly to balance the biogenesis and integrity of copper centers in vital enzymes against toxic levels of copper. PIB-type Cu+-ATPases play a central role in copper homeostasis by catalyzing the selective translocation of Cu+ across cellular membranes. Crystal structures of a copper-free Cu+-ATPase are available, but the mechanism of Cu+ recognition, binding, and translocation remains elusive. Through X-ray absorption spectroscopy, ATPase activity assays, and charge transfer measurements on solid-supported membranes using wild-type and mutant forms of the Legionella pneumophila Cu+-ATPase (LpCopA), we identify a sulfur-lined metal transport pathway. Structural analysis indicates that Cu+ is bound at a high-affinity transmembrane-binding site in a trigonal-planar coordination with the Cys residues of the conserved CPC motif of transmembrane segment 4 (C382 and C384) and the conserved Met residue of transmembrane segment 6 (M717 of the MXXXS motif). These residues are also essential for transport. Additionally, the studies indicate essential roles of other conserved intramembranous polar residues in facilitating copper binding to the high-affinity site and subsequent release through the exit pathway. 相似文献
5.
del Pino P Weiss A Bertsch U Renner C Mentler M Grantner K Fiorino F Meyer-Klaucke W Moroder L Kretzschmar HA Parak FG 《European biophysics journal : EBJ》2007,36(3):239-252
The cellular prion protein (PrPC) is a Cu2+ binding protein connected to the outer cell membrane. The molecular features of the Cu2+ binding sites have been investigated and characterized by spectroscopic experiments on PrPC-derived peptides and the recombinant human full-length PrPC (hPrP-[23-231]). The hPrP-[23-231] was loaded with 63Cu under slightly acidic (pH 6.0) or neutral conditions. The PrPC/Cu2+-complexes were investigated by extended X-ray absorption fine structure (EXAFS), electron paramagnetic resonance (EPR), and
electron nuclear double resonance (ENDOR). For comparison, peptides from the copper-binding octarepeat domain were investigated
in different environments. Molecular mechanics computations were used to select sterically possible peptide/Cu2+ structures. The simulated EPR, ENDOR, and EXAFS spectra of these structures were compared with our experimental data. For
a stoichiometry of two octarepeats per copper the resulting model has a square planar four nitrogen Cu2+ coordination. Two nitrogens belong to imidazole rings of histidine residues. Further ligands are two deprotonated backbone
amide nitrogens of the adjacent glycine residues and an axial oxygen of a water molecule. Our complex model differs significantly
from those previously obtained for shorter peptides. Sequence context, buffer conditions and stoichiometry of copper show
marked influence on the configuration of copper binding to PrPC.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
6.
James S. Hyde Brian Bennett Glenn L. Millhauser William E. Antholine 《Biophysical journal》2009,96(8):3354-3362
A double octarepeat prion protein construct, which has two histidines, mixed with copper sulfate in a 3:2 molar ratio provides at most three imidazole ligands to each copper ion to form a square-planar Cu2+ complex. This work is concerned with identification of the fourth ligand. A new (to our knowledge) electron paramagnetic resonance method based on analysis of the intense features of the electron paramagnetic resonance spectrum in the g⊥ region at 2 GHz is introduced to distinguish between three and four nitrogen ligands. The methodology was established by studies of a model system consisting of histidine imidazole ligation to Cu2+. In this spectral region at 2 GHz (S-band), g-strain and broadening from the possible rhombic character of the Zeeman interaction are small. The most intense line is identified with the MI = +1/2 extra absorption peak. Spectral simulation demonstrated that this peak is insensitive to cupric Ax and Ay hyperfine interaction. The spectral region to the high-field side of this peak is uncluttered and suitable for analysis of nitrogen superhyperfine couplings to determine the number of nitrogens. The spectral region to the low-field side of the intense extra absorption peak in the g⊥ part of the spectrum is sensitive to the rhombic distortion parameters Ax and Ay. Application of the method to the prion protein system indicates that two species are present and that the dominant species contains four nitrogen ligands. A new loop-gap microwave resonator is described that contains ∼1 mL of frozen sample. 相似文献
7.
Witkowska D Valensin D Rowinska-Zyrek M Karafova A Kamysz W Kozlowski H 《Journal of inorganic biochemistry》2012,107(1):73-81
The C-terminal region of Escherichia coli SlyD is unstructured and extremely rich in potential metal-binding amino acids, especially in histidine residues. SlyD is able to bind two to seven nickel ions per molecule, in a variety of coordination geometries and coordination numbers. This protein contributes to the insertion of nickel into the hydrogenase precursor protein and it has a peptidyl-prolyl cis/trans-isomerase activity which can be regulated through nickel ions. This inspired us to undertake systematic studies on the coordination ability of two histidine-rich peptides from the C-terminus of the SlyD protein with nickel. Also, it is known that histidine-rich regions are part of a Cu2 + binding domain involved in copper uptake under conditions of metal starvation in vivo in other bacteria. For this reason we decided to examine the complex formation of Ac-AHGHVHGAHDHHHD-NH2 and Ac-GHGHDHGHEHG-NH2 fragments with copper ions, which are also reference metal ions in this study. Experiments were performed in a DMSO/water 30:70 solvent. The Ac-AHGHVHGAHDHHHD-NH2 and Ac-GHGHDHGHEHG-NH2 fragments were synthesized and their interactions with Ni2 + and Cu2 + ions were studied by potentiometric, mass spectrometric, UV-vis, CD, EPR, and NMR spectroscopic techniques in solution. The results show that the Ac-GHGHDHGHEHG-NH2 fragment forms equimolar complexes with both nickel and copper ions. At physiological pH, the metal ion is bound only through nitrogens from imidazole sidechain of histidine residues. On the contrary, Ac-AHGHVHGAHDHHHD-NH2 binds 2 metal ions per molecule, at pH range 5 to 7, even if the 1:2 metal:peptide ratios were used. NMR studies indicate the involvement of all His residues in this pH-range in metal binding of the latter peptide. At higher pH, the stoichiometry changes to 1:1 and the His residues are displaced by amide nitrogens. 相似文献
8.
The prion protein is a ubiquitous neuronal membrane protein. Misfolding of the prion protein has been implicated in transmissible spongiform encephalopathies (prion diseases). It has been demonstrated that the human prion protein (PrP) is capable of coordinating at least five CuII ions under physiological conditions; four copper binding sites can be found in the octarepeat domain between residues 61 and 91, while another copper binding site can be found in the unstructured “amyloidogenic” domain between residues 91 and 126 PrP(91-126). Herein we expand upon a previous study [J. Shearer, P. Soh, Inorg. Chem. 46 (2007) 710-719] where we demonstrated that the physiologically relevant high affinity CuII coordination site within PrP(91-126) is found between residues 106 and 114. It was shown that CuII is contained within a square planar (N/O)3S coordination environment with one His imidazole ligand (H(111)) and one Met thioether ligand (either M(109) or M(112)). The identity of the Met thioether ligand was not identified in that study. In this study we perform a detailed investigation of the CuII coordination environment within the PrP fragment containing residues 106-114 (PrP(106-114)) involving optical, X-ray absorption, EPR, and fluorescence spectroscopies in conjunction with electronic structure calculations. By using derivatives of PrP(106-114) with systematic Met → Ile “mutations” we show that the CuII coordination environment within PrP(106-114) is actually comprised of a mixture of two major species; one CuII(N/O)3S center with the M(109) thioether coordinated to CuII and another CuII(N/O)3S center with the M(112) thioether coordinated to CuII. Furthermore, deletion of one or more Met residues from the primary sequence of PrP(106-114) both reduces the CuII affinity of the peptide by two to seven fold, and renders the resulting CuII metallopeptides redox inactive. The biological implications of these findings are discussed. 相似文献
9.
The native prion protein (PrP) has a two domain structure, with a globular folded α-helical C-terminal domain and a flexible
extended N-terminal region. The latter can selectively bind Cu2+ via four His residues in the octarepeat (OR) region, as well as two sites (His96 and His111) outside this region. In the
disease state, the folded C-terminal domain of PrP undergoes a conformational change, forming amorphous aggregates high in
β-sheet content. Cu2+ bound to the ORs can be redox active and has been shown to induce cleavage within the OR region, a process requiring conserved
Trp residues. Using computational modeling, we have observed that electron transfer from Trp residues to copper can be favorable.
These models also reveal that an indole-based radical cation or Cu+ can initiate reactions leading to protein backbone cleavage. We have also demonstrated, by molecular dynamics simulations,
that Cu2+ binding to the His96 and His111 residues in the remaining PrP N-terminal fragment can induce localized β-sheet structure,
allowing us to suggest a potential mechanism for the initiation of β-sheet misfolding in the C-terminal domain by Cu2+.
相似文献
Hans J. VogelEmail: |
10.
The metal ions Zn2+, Cu2+, and Fe2+ play a significant role in the aggregation mechanism of Aβ peptides. However, the nature of binding between metal and peptide has remained elusive; the detailed information on this from the experimental study is very difficult. Density functional theory (dft) (M06‐2X/6‐311++G (2df,2pd) +LANL2DZ) has employed to determine the force field resulting due to metal and histidine interaction. We performed 200 ns molecular dynamics (MD) simulation on Aβ1‐42‐Zn2+, Aβ1‐42‐Cu2+, and Aβ1‐42‐Fe2+ systems in explicit water with different combination of coordinating residues including the three Histidine residues in the N‐terminal. The present investigation, the Aβ1‐42‐Zn2+ system possess three turn conformations separated by coil structure. Zn2+ binding caused the loss of the helical structure of N‐terminal residues which transformed into the S‐shaped conformation. Zn2+ has reduced the coil and increases the turn content of the peptide compared with experimental study. On the other hand, the Cu2+ binds with peptide, β sheet formation is observed at the N‐terminal residues of the peptide. Fe2+ binding is to promote the formation of Glu22‐Lys28 salt‐bridge which stabilized the turn conformation in the Phe19‐Gly25 residues, subsequently β sheets were observed at His13‐Lys18 and Gly29‐Gly37 residues. The turn conformation facilitates the β sheets are arranged in parallel by enhancing the hydrophobic contact between Gly25 and Met35, Lys16 and Met35, Leu17 and Leu34, Val18 and Leu34 residues. The Fe2+ binding reduced the helix structure and increases the β sheet content in the peptide, which suggested, Fe2+ promotes the oligomerization by enhancing the peptide‐peptide interaction. Proteins 2016; 84:1257–1274. © 2016 Wiley Periodicals, Inc. 相似文献
11.
《Free radical research》2013,47(4):205-218
In our search to establish a reference ·OH production system with respect to which the reactivity of copper(II) complexes could then be tested, the influence of free Cu2+ ions on the Cu+/H2O2 reaction has been investigated.This influence depends on the CCu2+/CCu+ ratio. At low Cu2+ concentrations, ·OH damage to various detector molecules decreases with increasing Cu2+ concentrations until CCu2+/CCu+ reaches unity. Above this value, ·OH damage increases sharply until CCu2+/CCu+ becomes equal to 5 with salicylate and 2 with deoxyribose, ratios for which the protective effect of Cu2+ cancels. Finally, at higher concentrations, Cu2+ ions logically add their own ·OH production to that normally expected from Cu+ ions. The possible origin of this unprecedented alternate effect has been discussed. The possible influence of Cu+ ions on the generation of ·OH radicals by water gamma radiolysis has also been tested and, as already established for Cu2+ in a previous work, shown to be nonexistent. This definitely confirms that either form of ionised copper cannot scavenge ·OH radicals in the absence of a Iigand. 相似文献
12.
A novel multifunctional fluorescent peptide sensor based on pentapeptide dansyl‐Gly‐His‐Gly‐Gly‐Trp‐COOH (D‐P5) was designed and synthesized efficiently using Fmoc solid‐phase peptide synthesis (SPPS). This fluorescent peptide sensor shows selective and sensitive responses to Hg2+ and Cu2+ among 17 metal ions and six anions studied in N‐2‐hydroxyethylpiperazine‐N‐2‐ethane sulfonic acid (HEPES) buffer solution. The peptide probe differentiates Hg2+ and Cu2+ ions by a ‘turn‐on’ response to Hg2+ and a ‘turn‐off’ response to Cu2+. Upon addition of Hg2+ or Cu2+ ions, the sensor displayed an apparent color change that was visible under an ultraviolet lamp to the naked eye. The limits of detection (LOD) of DP‐5 were 25.0 nM for Hg2+ and 85.0 nM for Cu2+; the detection limits for Cu2+ were much lower than the drinking water maximum contaminant levels set out by the United States Environmental Protection Agency (USEPA). It is noteworthy that both D‐P5‐Hg and D‐P5‐Cu systems were also used to detect S2? successfully based on the formation of ternary complexes. The LODs of D‐P5‐Hg and D‐P5‐Cu systems for S2? were 217.0 nM and 380.0 nM, respectively. Furthermore, the binding stoichiometry, binding affinity and pH sensitivity of the probe for Hg2+ and Cu2+ were investigated. This study gives new possibilities for using a short fluorescent peptide sensor for multifunctional detection, especially for anions. 相似文献
13.
Inhibition by Cu2+ and Cd2+ of a mu‐class glutathione S‐transferase from shrimp Litopenaeus vannamei
Alex J. Salazar‐Medina Leticia García‐Rico Karina D. García‐Orozco Elisa Valenzuela‐Soto Carmen A. Contreras‐Vergara Rodrigo Arreola Aldo Arvizu‐Flores Rogerio R. Sotelo‐Mundo Ph.D. 《Journal of biochemical and molecular toxicology》2010,24(4):218-222
Glutathione S‐transferases (GSTs) are a family of detoxifying enzymes that catalyze the conjugation of glutathione (GSH) to electrophiles, thereby increasing the solubility of xenobiotics and aiding its excretion from the cell. The present work presents the inhibition of a mu‐class GST of the marine shrimp Litopenaeus vannamei by copper (Cu2+) and cadmium (Cd2+). The protein was overexpressed in bacteria and its enzymatic activity measured using 1‐chloro‐2,4‐dinitrobenzene. The mean inhibitory concentration (IC50) for shrimp GST against Cu2+ was 4.77 μM and for Cd2+ was 0.39 μM. A molecular model of the protein based on the crystal structure of a maize GST bound to cadmium showed that the metal binds in the GSH‐binding site by coordination with Asp and Gln residues. These results are consistent with the experimental data and suggest that sublethal concentration of metals may affect the capacity of the organism to detoxify pesticides or xenobiotics. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:218–222, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20326 相似文献
14.
蛋白质感染颗粒(PrP)的错误折叠被认为是引起一些神经退化性疾病的主因,但其正常构象(PrPC)的功能却一直不为人所知.近年来研究发现,在正常细胞中,尤其是脑细胞中,细胞膜PrPC可通过内吞作用进入细胞质而将Cu2+载运至SOD1,从而参与调节SOD1 的活性及细胞铜代谢.另有研究表明,Cu2+对于PrPSc(错误构象)的蛋白水解酶K抗性的恢复及不同“病株”的形成也有很重要的作用. 相似文献
15.
The effect of Cr2+ ions on the Tm (melting temperature) of DNA has been investigated under appropriate conditions for the stabilization of DNA by Mg2+ ions. A significant lowering of Tm, analogous to that observed for Cu2+ under normal conditions, was found, for Cr2+ at pH = 4.2 and [Mg2+] = 5.3 mol per mole of DNA base pair. Cu2+ also lowers Tm under similar conditions. The similarity of the effects of Cr2+ and Cu2+ under comparable conditions may be related to similarities in their coordination properties. It is proposed that Cr2+ and Cu2+ ions facilitate denaturation by holding together groups on the DNA chains in such a manner that base pairing and base stacking are inhibited. Comparative results for Cr3+ and Co2+ are also given for these low pH/Mg2+ ion conditions. 相似文献
16.
Chunnuan Deng Xiangliang Pan Shuzhi Wang Daoyong Zhang 《Biological trace element research》2014,160(2):268-275
Responses of photosystem I and II activities of Microcystis aeruginosa to various concentrations of Cu2+ were simultaneously examined using a Dual-PAM-100 fluorometer. Cell growth and contents of chlorophyll a were significantly inhibited by Cu2+. Photosystem II activity [Y(II)] and electron transport [rETRmax(II)] were significantly altered by Cu2+. The quantum yield of photosystem II [Y(II)] decreased by 29 % at 100 μg L?1 Cu2+ compared to control. On the contrary, photosystem I was stable under Cu2+ stress and showed an obvious increase of quantum yield [Y(I)] and electron transport [rETRmax(I)] due to activation of cyclic electron flow (CEF). Yield of cyclic electron flow [Y(CEF)] was enhanced by 17 % at 100 μg L?1 Cu2+ compared to control. The contribution of linear electron flow to photosystem I [Y(II)/Y(I)] decreased with increasing Cu2+ concentration. Yield of cyclic electron flow [Y(CEF)] was negatively correlated with the maximal photosystem II photochemical efficiency (F v/F m). In summary, photosystem II was the major target sites of toxicity of Cu2+, while photosystem I activity was enhanced under Cu2+ stress. 相似文献
17.
Visible circular dichroism (CD) spectra from the copper(II) titration of the metal-binding region of the prion protein, residues 57-98, were analyzed using the self-modeling curve resolution method multivariate curve resolution-alternating least squares (MCR-ALS). MCR-ALS is a set of mathematical tools for estimating pure component spectra and composition profiles from mixture spectra. Model-free solutions (e.g., soft models) are produced under the assumption that pure component profiles should be nonnegative and unimodal. Optionally, equality constraints can be used when the concentration or spectrum of one or more species is known. MCR-ALS is well suited to complex biochemical systems such as the prion protein which binds multiple copper ions and thus gives rise to titration data consisting of several pure component spectra with overlapped or superimposed absorption bands. Our study reveals the number of binding modes used in the uptake of Cu2+ by the full metal-binding region of the prion protein and their relative concentration profiles throughout the titration. The presence of a non-CD active binding mode can also be inferred. We show that MCR-ALS analysis can be initialized using empirically generated or mathematically generated pure component spectra. The use of small model peptides allows us to correlate specific Cu2+-binding structures to the pure component spectra. 相似文献
18.
Thyroglobulin (Tg) was subjected to metal-catalyzed oxidation, and the oxidative degradation was analyzed by SDS-polyacrylamide gel electrophoresis under reducing conditions. In contrast to no effect of hydrogen peroxide (H2O2) alone on the Tg degradation, the inclusion of Cu2+ (30 μM), in combination with 2 mM H2O2, caused a remarkable degradation of Tg, time- and concentration-dependent. The action of Cu2+ was not mimicked by Fe2+, suggesting that Tg may interact selectively with Cu2+. A similar degradation of Tg was also observed with Cu2+corbate system, and the concentration of Cu2+ (5–10 μM), in combination with ascorbate, required for the effective degradation was smaller than that of Cu2+ (10–30 μM) in combination with H2O2. In support of involvement of H2O2 in the Cu2+ corbate action, catalase expressed a complete protection. However, hydroxyl radical scavengers such as dimethylsulfoxide or mannitol failed to prevent the oxidation of Tg whereas phenolic compounds, which can interact with Cu2+, diminished the oxidative degradation, presumably consistent with the mechanism for Cu2+-catalyzed oxidation of protein. Moreover, the amount of carbonyl groups in Tg was increased as the concentration (3–100 μM) of Cu2+ was enhanced, while the formation of acid-soluble peptides was not remarkable in the presence of Cu2+ up to 200 μM. In further studies, Tg pretreated with heat or trichloroacetic acid seemed to be somewhat resistant to Cu2+-catalyzed oxidation, implying a possible involvement of protein conformation in the susceptibility to the oxidation. Based on these observations, it is proposed that Tg could be degraded non-enzymatically by Cu2+-catalyzed oxidation. 相似文献
19.
Jiang Zhou Junlei Hao Dajiang Wang Jiang Wu Zhongchang Wang Pengcheng Lin Jingcheng Hou 《Luminescence》2024,39(1):e4671
Copper is a critical element in both human and animal metabolic processes. Its role includes supporting connective tissue cross-linking, as well as iron and lipid metabolism; at the same time, copper is also a toxic heavy metal that can cause harm to both the environment and human health. Glutathione (GSH) is a tripeptide composed of glutamic acid, cysteine, and glycine combined with sulfhydryl groups. Its properties include acting as an antioxidant and facilitating integrative detoxification. GSH is present in both plant and animal cells and has a fundamental role in maintaining living organisms. GSH is the most abundant thiol antioxidant in the human body. It exists in reduced and oxidized forms within cells and provides significant biochemical functions, such as regulating vitamins such as vitamins D, E, and C, and facilitating detoxification. A fluorescent probe has been developed to detect copper ions selectively, sensitively, and rapidly. This report outlines the successful work on creating a peptide probe, TGN (TPE-Trp-Pro-Gly-Cln-His-NH2), with specific Cu2+ detection capabilities, and a significant fluorescence recovery occurred with the addition of GSH. This indicates that the probe can detect Cu2+ and GSH concurrently. The detection limit for Cu2+ in the buffer solution was 264 nM (R2 = 0.9992), and the detection limit for GSH using the TGN-Cu2+ complex was 919 nM (R2 = 0.9917). The probe exhibits high cell permeability and low biotoxicity that make it ideal for live cell imaging in biological conditions. This peptide probe has the capability to detect Cu2+ and GSH in biological cells. 相似文献
20.
Daniel Raimunda Poorna Subramanian Timothy Stemmler José M. Argüello 《生物化学与生物物理学报:生物膜》2012,1818(5):1374-1377
Zn2+ is an essential transition metal required in trace amounts by all living organisms. However, metal excess is cytotoxic and leads to cell damage. Cells rely on transmembrane transporters, with the assistance of other proteins, to establish and maintain Zn2+ homeostasis. Metal coordination during transport is key to specific transport and unidirectional translocation without the backward release of free metal. The coordination details of Zn2+ at the transmembrane metal binding site responsible for transport have now been established. Escherichia coli ZntA is a well-characterized Zn2+-ATPase responsible for intracellular Zn2+ efflux. A truncated form of the protein lacking regulatory metal sites and retaining the transport site was constructed. Metrical parameters of the metal–ligand coordination geometry for the zinc bound isolated form were characterized using x-ray absorption spectroscopy (XAS). Our data support a nearest neighbor ligand environment of (O/N)2S2 that is compatible with the proposed invariant metal coordinating residues present in the transmembrane region. This ligand identification and the calculated bond lengths support a tetrahedral coordination geometry for Zn2+ bound to the TM-MBS of P-type ATPase transporters. 相似文献