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Yeast metallothionein. Sequence and metal-binding properties 总被引:31,自引:0,他引:31
D R Winge K B Nielson W R Gray D H Hamer 《The Journal of biological chemistry》1985,260(27):14464-14470
The protein product of the CUP1 locus in Cu-resistant Saccharomyces cerevisiae has been purified and characterized. The protein was found to lack the first 8 amino acids predicted by the nucleotide sequence of the gene. The residues removed from the amino-terminal region include 5 hydrophobic residues, two of which are aromatic. The unique amino terminus starting at Gln9 of the putative DNA translation product was observed for metallothionein purified in the presence of various protease inhibitors or from a pep4 mutant yeast strain deficient in vacuolar proteases. The remainder of the primary structure of the protein is equivalent to the decoded DNA sequence, so yeast metallothionein is a 53-residue polypeptide of molecular weight 5655. The isolated protein contained 8 copper ions ligated by 12 cysteines/molecule. Reconstitution studies of the apo-molecule revealed that 8 mol eq of Cu(I) conferred maximal stability against proteolysis and depleted the zinc content of zinc-saturated metallothionein. These assays suggested that the protein has 8 binding sites for Cu(I). Ag(I) ions bound to the protein with the same stoichiometry. Yeast metallothionein was also observed to coordinate Cd(II) and Zn(II) ions in vitro. In studies of direct binding, protection against proteolysis, and metal ion exchange, these divalent ions were found to associate with the protein with a maximal stoichiometry of 4 ions/molecule. Yeast metallothionein thus exhibits two distinct binding configurations for Cu(I) and Cd(II) as does the mammalian protein. 相似文献
3.
M Shoham A Yonath J L Sussman J Moult W Traub A J Kalb 《Journal of molecular biology》1979,131(2):137-155
We have determined the crystal structure of demetallized concanavalin A, at a resolution of 3.2 Å, by molecular replacement using the known structure of native concanavalin A. Refinement of the initial model using a constraint-restraint reciprocal-space least-squares procedure caused the conventional crystallographic agreement (R) factor to decrease from 0.47 to a final value of 0.26. There are significant conformational changes in the metal-binding region involving residues Asp 19 and His24, which are substantially closer to each other than in native concanavalin A. These residues form an internal salt bridge which does not exist when the metal ions are attached to the protein. The binding site for transitionmetal ions is still intact, but the calcium site is not, since one of its two carboxylic ligands, Asp 19, is unavailable. Flexibility is observed for one of the transitionmetal ligands, Glu8, as well as for some segments of the backbone. The latter could account for the increased susceptibility of demetallizcd concanavalin A to proteolysis. 相似文献
4.
G Gutzke B Fischer R R Mendel G Schwarz 《The Journal of biological chemistry》2001,276(39):36268-36274
Molybdopterin (MPT) is a pyranopterin with a unique dithiolene group coordinating molybdenum (Mo) or tungsten (W) in all Mo- and W-enzymes except nitrogenase. In Escherichia coli, MPT is formed by incorporation of two sulfur atoms into precursor Z, which is catalyzed by MPT synthase. The recently solved crystal structure of MPT synthase (Rudolph, M. J., Wuebbens, M. M., Rajagopalan, K. V., and Schindelin, H. (2000) Nat. Struct. Biol. 8, 42-46) shows the heterotetrameric nature of the enzyme that is composed of two small (MoaD) and two large subunits (MoaE). According to sequence and structural similarities among MoaD, ubiquitin, and ThiS, a thiocarboxylation of the C terminus of MoaD is proposed that would serve as the source of sulfur that is transferred to precursor Z. Here, we describe the in vitro generation of carboxylated and thiocarboxylated MoaD. Both forms of MoaD are monomeric and are able to form a heterotetrameric complex after coincubation in equimolar ratios with MoaE. Only the thiocarboxylated MPT synthase complex was found to be able to convert precursor Z in vitro to MPT. Slight but significant differences between the carboxylated and the thiocarboxylated MPT synthase can be seen using size exclusion chromatography. A two-step reaction of MPT synthesis is proposed where the dithiolene is generated by two thiocarboxylates derived from a single tetrameric MPT synthase. 相似文献
5.
Human nucleotide excision repair protein XPA: extended X-ray absorption fine-structure evidence for a metal-binding domain. 总被引:3,自引:0,他引:3
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N. J. Hess G. W. Buchko S. D. Conradson F. J. Espinosa S. Ni B. D. Thrall M. A. Kennedy 《Protein science : a publication of the Protein Society》1998,7(9):1970-1975
The ubiquitous, multi-enzyme, nucleotide excision repair (NER) pathway is responsible for correcting a wide range of chemically and structurally distinct DNA lesions in the eukaryotic genome. Human XPA, a 31 kDa, zinc-associated protein, is thought to play a major NER role in the recognition of damaged DNA and the recruitment of other proteins, including RPA, ERCC1, and TFIIH, to repair the damage. Sequence analyses and genetic evidence suggest that zinc is associated with a C4-type motif, C105-X2-C108-X17-C126-X2-C129, located in the minimal DNA binding region of XPA (M98-F219). The zinc-associated motif is essential for damaged DNA recognition. Extended X-ray absorption fine structure (EXAFS) spectra collected on the zinc associated minimal DNA-binding domain of XPA (ZnXPA-MBD) show directly, for the first time, that the zinc is coordinated to the sulfur atoms of four cysteine residues with an average Zn-S bond length of 2.34+/-0.01 A. XPA-MBD was also expressed in minimal medium supplemented with cobalt nitrate to yield a blue-colored protein that was primarily (>95%) cobalt associated (CoXPA-MBD). EXAFS spectra collected on CoXPA-MBD show that the cobalt is also coordinated to the sulfur atoms of four cysteine residues with an average Co-S bond length of 2.33+/-0.02 A. 相似文献
6.
I Bertini C Luchinat L Messori R Monnanni A Scozzafava 《The Journal of biological chemistry》1986,261(3):1139-1146
Cobalt(II) ovotransferrin bicarbonate and oxalate ternary complexes were prepared and investigated in the pH range 7-10.5. Cobalt(II) provides an excellent and unique spectroscopic probe to monitor subtle structural differences in solution between the two sites of ovotransferrin and to investigate the structural dependence on pH. CD spectroscopy on one side and 1H NMR spectroscopy of isotropically shifted signals on the other are extremely sensitive techniques and are particularly suited for high spin cobalt(II)-containing compounds. In the case of the oxalate derivative the metal-binding ability of the protein is different at the two binding sites and is pH dependent; the CD spectra reveal two different sites, one of which is clearly pH dependent with a pKa of 9.5. On the contrary the bicarbonate analogue does not show any spectral difference between the two sites; both of them change with pH, the pKa being again 9.5. 1H NMR spectra of the oxalate derivatives at pH 7-8 reveal the presence of conformers, the distribution of which depends on the H2O/D2O ratio. Such conformers are not revealed in the bicarbonate system; at pH around 10 the NMR spectra of both systems show inequivalence between the two sites and/or the presence of different conformers for each site. Such differences are discussed in terms of the possible implications in mechanism and function. The overall spectral data are consistent with the donor groups being two histidines, two tyrosines, the synergistic anion, and possibly a solvent molecule. 相似文献
7.
Changes in cardiac contractility related to calcium-mediated changes in phosphorylation of myosin-binding protein C.
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Ca ions can influence the contraction of cardiac muscle by activating kinases that specifically phosphorylate the myofibrillar proteins myosin-binding protein C (MyBP-C) and the regulatory light chain of myosin (RLC). To investigate the possible role of Ca-regulated phosphorylation of MyBP-C on contraction, isolated quiescent and rhythmically contracting cardiac trabeculae were exposed to different concentrations of extracellular Ca and then chemically skinned to clamp the contractile system. Maximum Ca-activated force (F(max)) was measured in quiescent cells soaking in 1) 2.5 mM Ca for 120 min, 2) 1.25 mM for 120 min, or 3) 1.25 mM for 120 min followed by 10 min in 7.5 mM, and 4) cells rhythmically contracting in 2.5 mM for 20 min. F(max) was, respectively, 21.5, 10.5, 24.7, and 32.6 mN/mm(2). Changes in F(max) were closely associated with changes in the degree of phosphorylation of MyBP-C and occurred at intracellular concentrations of Ca below levels associated with phosphorylation of RLC. Monophosphorylation of MyBP-C by a Ca-regulated kinase is necessary before beta-adrenergic stimulation can produce additional phosphorylation. These results suggest that Ca-dependent phosphorylation of MyBP-C modulates contractility by changing thick filament structure. 相似文献
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D C Harris 《Biochemistry》1977,16(3):560-564
Transferrin, the serum serum iron-transport protein which can bind two metal ions at physiologic pH, binds just one Fe3+, VO2+, or Cr3+ ion at pH 6.0. Fe3+ and VO2+ appear to be bound at the same site, designated A, based on electron paramagnetic resonance (EPR) spectra of VO2+-transferrin and (Fe3+)1(VO2+)1-transferrin. The EPR spectra of (Cr3+)1(VO2+)1-transferrin and of (Cr3+), (FE3+)1-transferrin indicate that that Cr3+ is bound to site B at pH 6.0. Transferrin was labeled at site A with 59Fe at pH 6.0 and at site B with 55Fe at pH 7.5. When the pH of the resulting preparation was lowered to 6.3 and the dissociated iron was separated by gel filtration, about ten times as much 55Fe as 59Fe was lost. The same EPR and isotopic-labeling experiments showed that Fe3+ added to transferrin at pH 7.5 binds to site A with about 90% selectivity. 相似文献
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C. Henderson 《HOMO》2013
Changes in subsistence strategy have caused some of the profoundest changes to the structure and health of humans. This study aims to test whether these changes have reduced work-load as assessed by entheseal changes. Entheseal changes, formerly called musculoskeletal stress markers, are thought to reflect muscle usage throughout life, although it is widely agreed that they have a multifactorial origin. This paper uses a meta-analysis of comparable published data to plot trends in time by muscle, enthesis type and sex. The results show that agriculturalists have the lowest scores for entheseal changes, with hunte–gatherers next highest and those working in industry the highest. These findings are the same for males and females, for most muscles and muscle groups. However, entheseal changes are highly correlated with increased age and the age distributions of the samples analysed could not be compared. It is, therefore, possible that differences in age distribution of the samples are one of the reasons for this finding. Recommendations are provided to reduce this and other limitations for future meta-analyses. 相似文献
10.
J S D'Arrigo 《The Journal of membrane biology》1975,22(3-4):255-263
The effect of different extracellular alkaline-earth cations (Ca2+, Mg2+, Sr2+, Ba2+) upon the threshold membrane potential for spike initiation in crayfish axon has been studied by means of intracellular microelectrodes. This was done at the following extracellular concentrations of the divalent uranyl ion (UO2/2+): 1.0 X 10(-6) M, 3.0 X 10(-6) M, and 9.0 X 10(-6) M. At each concentration employed, extensive neutralization of axonal surface charges by UO2/2+ was evidenced by the fact that equal concentrations (50 mM) of alkaline-earth cations did not have the same effect on the threshold potential. The selectivity sequences observed at the different uranyl-ion concentrations were: 1.0 X 10(-6) M UO2/2+, Ca2+ greater than Mg2+ greater than Sr2+ greater than Ba2+; 3.0 X 10(-6) M UO2/2+, Ca2+ greater than Mg2+ greater than Ba2+ larger than or equal to Sr2+; 9.0 X 10(-6) M UO2/2+, Ca2+ approximately Ba2+ greater than Sr2+ greater than Mg2+. These selectivity sequences are in accord with the equilibrium selectivity theory for alkaline-earth cations. At each of the concentrations used, uranyl ion did not have any detectable effect on the actual shape of the action potential itself. It is concluded that many (if not most) of the surface acidic groups in the region of the sodium gates represent phosphate groups of membrane phospholipids, but that the m gates themselves are probably protein-aceous in structure. 相似文献
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P Dahiya M Kumbhakar D K Maity T Mukherjee J P Mittal A B R Tripathi N Chattopadhyay H Pal 《Photochemical & photobiological sciences》2005,4(1):100-105
The photophysical properties of 2-amino-9,10-anthraquinone (2AAQ) have been investigated in different solvents and solvent mixtures and correlated with the Lippert-Mataga solvent polarity parameter, Deltaf. In the low solvent polarity region with Deltaf < ca. 0.1, the dye shows unusually high fluorescence quantum yields (Phif) and lifetimes (tauf) in comparison to those in other solvents of medium to high polarities. Similarly, the radiative rate constants (kf) are relatively lower and the non-radiative rate constants (knr) are relatively higher in the low polarity solvents in comparison to those in the medium to high polarity solvents. The current results have been rationalized assuming that the dye adopts different structural forms below and above the Deltaf value of approximately 0.1. It is inferred that in the low solvent polarity region the dye exists in a non-planar structure, with its 2-NH2 plane away from that of the 9,10-anthraquinone moiety in the ground state. In solvents of medium to high polarities, the dye exists in a polar intramolecular charge transfer (ICT) structure, where the amino lone pair of the 2-NH2 group is in strong resonance with the anthraquinone pi-cloud in the ground state. In all the solvents, however the dye is inferred to exist in the ICT structure in its excited (S1) state. Supportive evidence for the above hypothesis has been obtained from the solvent polarity effect on the Stokes' shifts for the dye. Quantum chemical studies on the structures of 2AAQ dye in the gas phase also give qualitative support for the inferences drawn from the photophysical properties of the dye in different solvents. 相似文献
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The addition of Mn2+, Zn2+, Co2+, Ca2+ or Pb2+ to apo-concanavalin A results in a slow conformational conversion of the protein to the active saccharide binding form. The rates of conversion are dependent upon the sample pH and identity of the ions which occupy the native transition metal and calcium ion sites yet the affinity of each metalloform for the fluorescent sugar, 4-methylumbelliferyl-α-D-mannopyranoside, is independent of these same parameters (above pH 5.6). EDTA quickly removes all metal ions from the active Mn2+ or Co2+-concanavalin A samples leaving a metastable metal free structure which retains its high saccharide affinity for several hours at room temperature. This form of apo-concanavalin A and the metallized derivatives have equally high saccharide binding affinities in 1M NaCL but the former dramatically loses its sugar affinity as the ionic strength is lowered. 相似文献
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Electrospray ionization mass spectrometry of zinc, cadmium, and copper metallothioneins: evidence for metal-binding cooperativity 总被引:3,自引:0,他引:3
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Gehrig PM You C Dallinger R Gruber C Brouwer M Kägi JH Hunziker PE 《Protein science : a publication of the Protein Society》2000,9(2):395-402
Electrospray ionization (ESI) mass spectra of both well-characterized and novel metallothioneins (MTs) from various species were recorded to explore their metal-ion-binding modes and stoichiometries. The ESI mass spectra of the zinc- and cadmium-binding MTs showed a single main peak corresponding to metal-to-protein ratios of 4, 6, or 7. These findings combined with data obtained by other methods suggest that these MTs bind zinc or cadmium in a single predominant form and are consistent with the presence of three- and four-metal clusters. An unstable copper-specific MT isoform from Roman snails (Helix pomatia) could be isolated intact and was shown to preferentially bind 12 copper ions. To obtain additional information on the formation and relative stability of metal-thiolate clusters in MTs, a mass spectrometric titration study was conducted. One to seven molar equivalents of zinc or of cadmium were added to metal-free human MT-2 at neutral pH, and the resulting complexes were measured by ESI mass spectrometry. These experiments revealed that the formation of the four-metal cluster and of the thermodynamically less stable three-metal cluster is sequential and largely cooperative for both zinc and cadmium. Minor intermediate forms between metal-free MT, Me4MT, and fully reconstituted Me7MT were also observed. The addition of increasing amounts of cadmium to metal-free blue crab MT-I resulted in prominent peaks whose masses were consistent with apoMT, Cd3MT, and Cd6MT, reflecting the known structure of this MT with two Me3Cys9 centers. In a similar reconstitution experiment performed with Caenorhabditis elegans MT-II, a series of signals corresponding to apoMT and Cd3MT to Cd6MT species were observed. 相似文献
15.
R M Cooke B G Carter P Murray-Rust M J Hartshorn P Herzyk R E Hubbard 《Protein engineering》1992,5(6):473-477
The solution structure of the fibrinogen antagonist, echistatin, has been determined by a combination of NMR and simulated annealing methods. While the structure of the disulphide-linked core is well-defined by the NMR data, the N- and C-termini and the loop bearing the RGD sequence (which is responsible for the fibrinogen antagonist properties) are poorly defined. The pattern of disulphide bridges, which could not be determined by classical methods, was predicted by a statistical analysis of the simulated annealing structures. This pattern is distinct from that for the homologous protein kistrin, leading to the novel suggestion that homologous proteins possess non-conserved patterns of disulphide bridges. 相似文献
16.
Isotope labeling of recombinant normal cardiac troponin C (cTnC3) with 15N-enriched amino acids and multidimensional NMR were used to assign the downfield-shifted amide protons of Gly residues at position 6 in Ca(2+)-binding loops II, III, and IV, as well as tightly hydrogen-bonded amides within the short antiparallel beta-sheets between pairs of Ca(2+)-binding loops. The amide protons of Gly70, Gly110, and Gly146 were found to be shifted significantly downfield from the remaining amide proton resonances in Ca(2+)-saturated cTnC3. No downfield-shifted Gly resonance was observed from the naturally inactive site I. Comparison of downfield-shifted amide protons in the Ca(2+)-saturated forms of cTnC3 and CBM-IIA, a mutant having Asp65 replaced by Ala, demonstrated that Gly70 is hydrogen bonded to the carboxylate side chain of Asp65. Thus, the hydrogen bond between Gly and Asp in positions 6 and 1, respectively, of the Ca(2+)-binding loop appears crucial for maintaining the integrity of the helix-loop-helix Ca(2+)-binding sites. In the apo- form of cTnC3, only Gly70 was found to be shifted significantly downfield with respect to the remaining amide proton resonances. Thus, even in the absence of Ca2+ at binding site II, the amide proton of Gly70 is strongly hydrogen bonded to the side-chain carboxylate of Asp65. The amide protons of Ile112 and Ile148 in the C-terminal domain and Ile36 in the N-terminal domain data-sheets exhibit chemical shifts consistent with hydrogen-bond formation between the pair of Ca(2+)-binding loops in each domain of Ca(2+)-saturated cTnC3.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
17.
Protamine-like (PL) proteins are DNA-condensing proteins that replace somatic-type histones during spermatogenesis. Their composition suggests a function intermediate to that of histones and protamines. Although these proteins have been well characterized at the chemical level in a large number of species, particularly in marine invertebrates, little is known about the specific structures arising from their interaction with DNA. Speculation concerning chromatin structure is complicated by the high degree of heterogeneity in both the number and size of these proteins, which can vary considerably even between closely related species. After careful examination and comparison of the protein sequences available to date for the PL proteins, we propose a model for a novel chromatin structure in the sperm of these organisms that is mediated by somatic-type histones, which are frequently found associated with these proteins. This structure supports the concept that the PL proteins may represent various evolutionary steps between a sperm-specific histone H1 precursor and true protamines. Potential post-translational modifications and the control of PL protein expression and deposition are also discussed. 相似文献
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To investigate the metal-binding properties of KChIP1, the interaction of KChIP1 and mutated KChIP1 with divalent cations (Mg(2+), Ca(2+), Sr(2+), and Ba(2+)) was explored by 8-anilinonaphthalene-1-sulfonate (ANS) fluorescence. It showed that KChIP1 possessed two types of Ca(2+)-binding sites, high-affinity and low-affinity Ca(2+)-binding sites. However, only low-affinity-binding site for Mg(2+), Sr(2+), and Ba(2+) was observed. The metal-binding properties of KChIP1 are not appreciably affected after removal of the N-terminal portion and EF-hand 1. Deleting the EF-hand 4 of KChIP1 abolishes its high-affinity Ca(2+)-binding site, but retains the intact low-affinity-binding site for metal ions. A decrease in the nonpolarity of ANS-binding site occurs with all mutants. However, the binding of ANS with KChIP1 is no longer observed after removal of EF-hands 3 and 4. Intermolecular interaction assessed by chemical cross-linking suggested that KChIP1 had a propensity to form dimer in the absence of metal ions, and a KChIP1 tetramer was pronouncedly produced in the presence of metal ions. Noticeably, the oligomerization state depends on the integrity of EF-hand 4. Taken together, our data suggest that EF-hand 4 is of structural importance as well as functional importance for fulfilling the physiological function of KChIP1. 相似文献