共查询到20条相似文献,搜索用时 78 毫秒
1.
Divalent Cations Crosslink Vimentin Intermediate Filament Tail Domains to Regulate Network Mechanics
Yi-Chia Lin Chase P. Broedersz Tatjana Wedig Frederick C. MacKintosh 《Journal of molecular biology》2010,399(4):637-1289
Intermediate filament networks in the cytoplasm and nucleus are critical for the mechanical integrity of metazoan cells. However, the mechanism of crosslinking in these networks and the origins of their mechanical properties are not understood. Here, we study the elastic behavior of in vitro networks of the intermediate filament protein vimentin. Rheological experiments reveal that vimentin networks stiffen with increasing concentrations of Ca2+ and Mg2+, showing that divalent cations act as crosslinkers. We quantitatively describe the elastic response of vimentin networks over five decades of applied stress using a theory that treats the divalent cations as crosslinkers: at low stress, the behavior is entropic in origin, and increasing stress pulls out thermal fluctuations from single filaments, giving rise to a nonlinear response; at high stress, enthalpic stretching of individual filaments significantly modifies the nonlinearity. We investigate the elastic properties of networks formed by a series of protein variants with stepwise tail truncations and find that the last 11 amino acids of the C-terminal tail domain mediate crosslinking by divalent ions. We determined the single-filament persistence length, lP ≈ 0.5 μm, and Young's modulus, Y ≈ 9 MPa; both are consistent with literature values. Our results provide insight into a crosslinking mechanism for vimentin networks and suggest that divalent ions may help regulate the cytoskeletal structure and mechanical properties of cells. 相似文献
2.
The voltage-gated proton channel Hv1 functions as a dimer, in which the intracellular C-terminal domain of the protein is responsible for the dimeric architecture and regulates proton permeability. Although it is well known that divalent metal ions have effect on the proton channel activity, the interaction of divalent metal ions with the channel in detail is not well elucidated. Herein, we investigated the interaction of divalent metal ions with the C-terminal domain of human Hv1 by CD spectra and fluorescence spectroscopy. The divalent metal ions binding induced an obvious conformational change at pH 7 and a pH-sensitive reduction of thermostability in the C-terminal domain. The interactions were further estimated by fluorescence spectroscopy experiments. There are at least two binding sites for divalent metal ions binding to the C-terminal domain of Hv1, either of which is close to His244 or His266 residue. The binding of Zn2+ to the two sites both enhanced the fluorescence of the protein at pH 7, whereas the binding of other divalent metal ions to the two sites all resulted fluorescence quenching. The orders of the strength of divalent metal ions binding to the two sites from strong to weak are both Co2+, Ca2+, Ni2+, Mg2+, and Mn2+. The strength of Ca2+, Co2+, Mg2+, Mn2+ and Ni2+ binding to the site close to His244 is stronger than that of these divalent metal ions binding to the site close to His266. 相似文献
3.
Characterization of the metal ion binding site in the anti-terminator protein, HutP, of Bacillus subtilis 总被引:1,自引:0,他引:1
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
HutP is an RNA-binding protein that regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis, by binding to cis-acting regulatory sequences on hut mRNA. It requires L-histidine and an Mg2+ ion for binding to the specific sequence within the hut mRNA. In the present study, we show that several divalent cations can mediate the HutP–RNA interactions. The best divalent cations were Mn2+, Zn2+ and Cd2+, followed by Mg2+, Co2+ and Ni2+, while Cu2+, Yb2+ and Hg2+ were ineffective. In the HutP–RNA interactions, divalent cations cannot be replaced by monovalent cations, suggesting that a divalent metal ion is required for mediating the protein–RNA interactions. To clarify their importance, we have crystallized HutP in the presence of three different metal ions (Mg2+, Mn2+ and Ba2+), which revealed the importance of the metal ion binding site. Furthermore, these analyses clearly demonstrated how the metal ions cause the structural rearrangements that are required for the hut mRNA recognition. 相似文献
4.
The effects of various metal ions on cleavage activity and global folding have been studied in the extended Schistosoma hammerhead ribozyme. Fluorescence resonance energy transfer was used to probe global folding as a function of various monovalent and divalent metal ions in this ribozyme. The divalent metals ions Ca2+, Mg2+, Mn2+, and Sr2+ have a relatively small variation (less than sixfold) in their ability to globally fold the hammerhead ribozyme, which contrasts with the very large difference (>10,000-fold) in apparent rate constants for cleavage for these divalent metal ions in single-turnover kinetic experiments. There is still a very large range (>4600-fold) in the apparent rate constants for cleavage for these divalent metal ions measured in high salt (2 M NaCl) conditions where the ribozyme is globally folded. These results demonstrate that the identity of the divalent metal ion has little effect on global folding of the Schistosoma hammerhead ribozyme, whereas it has a very large effect on the cleavage kinetics. Mechanisms by which the identity of the divalent metal ion can have such a large effect on cleavage activity in the Schistosoma hammerhead ribozyme are discussed. 相似文献
5.
The mechanical properties of epithelial cells are modulated by structural changes in keratin intermediate filament networks. To investigate the relationship between network architecture and viscoelasticity, we assembled keratin filaments from recombinant keratin proteins 8 (K8) and 18 (K18) in the presence of divalent ions (Mg2+). We probed the viscoelastic modulus of the network by tracking the movement of microspheres embedded in the network during assembly, and studied the network architecture using scanning electron microscopy. Addition of Mg2+ at physiological concentrations (<1 mM) resulted in networks whose structure was similar to that of keratin networks in epithelial cells. Moreover, the elastic moduli of networks assembled in vitro were found to be within the same magnitude as those measured in keratin networks of detergent-extracted epithelial cells. These findings suggest that Mg2+-induced filament cross-linking represents a valid model for studying the cytoskeletal mechanics of keratin networks. 相似文献
6.
Divalent metal ions promote hydrolysis of RNA backbones generating 5′OH and 2′;3′P as cleavage products. In these reactions, the neighboring 2′OH act as the nucleophile. RNA catalyzed reactions also require divalent metal ions and a number of different metal ions function in RNA mediated cleavage of RNA. In one case, the LZV leadzyme, it was shown that this catalytic RNA requires lead for catalysis. So far, none of the naturally isolated ribozymes have been demonstrated to use lead to activate the nucleophile. Here we provide evidence that RNase P RNA, a naturally trans-acting ribozyme, has leadzyme properties. But, in contrast to LZV RNA, RNase P RNA mediated cleavage promoted by Pb2+ results in 5′ phosphate and 3′OH as cleavage products. Based on our findings, we infer that Pb2+ activates H2O to act as the nucleophile and we identified residues both in the substrate and RNase P RNA that most likely influenced the positioning of Pb2+ at the cleavage site. Our data suggest that Pb2+ can promote cleavage of RNA by activating either an inner sphere H2O or a neighboring 2′OH to act as nucleophile. 相似文献
7.
Surface potentials of phosphatidylserine monolayers have been measured in the presence of different divalent ion concentrations in order to determine the way in which divalent ions bind to the membrane surface. The association constants for divalent ions (Mg2+, Ca2+ and Mn2+) with the phosphatidylserine membrane have been obtained from the experimental data and simple ion binding theory. The order of divalent ion binding to the membrane is Mn2+ > Ca2+ > Mg2+. However, none of the divalent ions used completely neutralized the negative charge of phosphatidylserine even at relatively high concentrations. The amounts of the divalent ions bound depended upon the concentration of the monovalent ions present in the subphase. It is suggested that the amounts of bound ions obtained from the use of radioisotope tracer methods may include a considerable contribution from the excess free ions in the double layer region of the phosphatidylserine membrane. 相似文献
8.
The divalent metal ions Cu2+, Co2+, Mn2+, and Zn2+ form complexes with the fluorescent etheno analogs of the adenine nucleotides. The fluorescence intensity is thereby diminished. The binding strength of the metals to etheno-adenosine triphosphate is higher than to etheno-adenosine di- and monophosphate. The quenching effect of the divalent metal ions can be exploited as a simple routine activity measurement for various kinases and phosphohydrolases. 相似文献
9.
Electrophoretic mobility and 31P NMR measurements were combined to test whether the combination of the Henry, Boltzmann and Grahame equations is capable of describing the adsorption of divalent cations to phosphatidylcholine membranes. Cobalt was chosen for this study because, of all the common divalent cations, its effects on the 31P NMR spectrum of phosphatidylcholine membranes are easiest to interpret. Both the 31P NMR data on the adsorption of cobalt and the zeta potential data calculated from the electrophoretic mobility in the presence of cobalt are well described by the combination of these three equations. Electrophoretic mobility measurements were also performed with a number of other divalent cations and the zeta potentials were, in all cases, well described by the combination of these three equations. The binding deduced from such measurements decreases in the sequence: Mn2+, Mg2+, Ca2+, Co2+, Ni2+, Sr2+, Ba2+. If we assume that a lipid molecule occupies an area of 60 Å2 and that there is a 1: 1 stoichiometry for the binding of the divalent ions to phosphatidylcholine, the dissociation constants are, respectively: 0.3, 1.0, 1.0, 1.2, 1.2, 2.8, 3.6 M. 相似文献
10.
P2X2 purinoceptors are cation-selective channels activated by ATP and its analogues. Using single channel measurements we studied
the channel's selectivity for the alkali metal ions and organic monovalent cations NMDG+, Tris+, TMA+, and TEA+. The selectivity sequence for currents carried by alkali metal ions is: K+ > Rb+ > Cs+ > Na+ > Li+, which is Eisenman sequence IV. This is different from the mobility sequence of the ions in free solution suggesting there is weak interaction
between the ions and the channel interior. The relative conductance for alkali ions increases linearly in relation to the
Stokes radius. The organic ions NMDG+, Tris+, TMA+ and TEA+ were virtually impermeant. The divalent ions (Mn2+, Mg2+, Ca2+ and Ba2+) induced a fast block visible as a reduction in amplitude of the unitary currents. Using a single-site binding model, the
divalent ions exhibited an equilibrium affinity sequence of Mn2+ > Mg2+ > Ca2+ > Ba2+.
Received: 3 May 1999/Revised: 23 August 1999 相似文献
11.
Nelwyn T. Christie R.K. Owenby K.Bruce Jacobson V.S. Hiatt W.R. Farkas 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1982,699(1):40-48
Dietary cadmium causes the queuine-containing, Q(+), isoacceptors to increase relative to the guanine-containing, Q(?), ones of tRNATyr, tRNAHis and tRNAAsp of Drosophila melanogaster. Of the other divalent cations examined, Sr2+, Ni2+, Cu2+, Zn2+ and Hg2+, only Hg2+ failed to cause an increase in Q(+)tRNATyr. For these results, all pre-adult stages of the organism were spent on media containing the divalent ions. Adult flies that had developed on a normal diet also responsed to divalent ions; Hg2+ as well as Cd2+, Sr2+ and Zn2+ caused an increase in Q(+)tRNATyr in 4 days. Using adult flies, the rate of the response was measured; when placed on a Cd2+-containing diet, they formed significantly more Q(+)tRNATyr within 24 h as compared to adults on a normal diet. Whether the queuine is derived from the diet or from de novo synthesis is yet to be determined. Since the metal ions represent a range of values in the ‘hard-soft’ classification, different sites of reaction are expected, yet for Drosophila a common result is an alteration in the ratio of Q(+) and Q(?) isoacceptors of these tRNAs. The transition to Q(+)tRNA may be an early indication of the metabolic imbalances resulting from the presence of the divalent cation. 相似文献
12.
Axoplasm extruded from the giant axon of the squid contains Ca2+-activated proteases. The protease in the 100,000X g of supernatant of axoplasm is very specific and degrades only the 200,000 MW, neurofilament protein (NF200), whereas the protease(s) in the pellet has a much wider range of substrate specificity. The activation of the supernatant protease is restricted to the Ca2+ ion, and no other divalent cation will substitute. The protease requires Ca2+ at a higher concentration than 0.5 mM for activation, and has a pH optimum of about 7.5. Degradation of the NF200 appears to proceed through a 100,000 MW and possibly a 47,000–50,000-MW intermediate form before degradation to TCA-soluble peptides. Activity of the protease is inhibited by divalent cation chelators, Cu2+ and Fe2+, sulphydryl inhibitors, and leupeptin. This specific Ca2+-activated protease in squid axoplasm has identical properties to Ca2+-activated proteases found in various non-neural tissues. Despite its narrow protein substrate specificity, Ca2+-activated protease purified from human platelets effectively degrades squid NF200, suggesting a possible structural relationship between platelet and muscle actin-binding proteins and neurofilament proteins. 相似文献
13.
Calzuola Isabella Gianfranceschi Gian Luigi Marsili Valeria 《Molecular biology reports》2001,28(1):43-46
Citric acid whose structure is comparable to that of small acidic peptides, can bind to DNA in the presence of divalent cations (Cu2+, Fe2+, Zn2+, Mg2+). Citrate-DNA interaction occurs also in a cell homogenate and in this experimental model too requires the presence of natural divalent cations. In fact the addition of 2 mM EDTA to cell homogenate strongly decreases the DNA-citrate binding. The results demonstrate that divalent cations can act as bridges between two acidic molecules and that citric acid can mimic the structure of acidic peptides. 相似文献
14.
Chiroptical properties of heparin for various degrees of neutralization of the sulfate and carboxylic groups and for different counter-ions in salt-free aqueous solutions were investigated. Variations of optical rotation and ellipticity values at given wavelengths are compared to simultaneous pH and viscosity changes observed during the neutralization of heparin by sodium and calcium hydroxide. For Na+, variations of ellipticity at 210 nm are related to acid—base properties of uronic carboxylic groups. C.d. characteristics found for alkaline-earth counter-ions (Mg2+, Ca2+ and Ba2+), as compared to Na+, are assigned to effects of divalent ions on the ionization behavior of carboxylic groups. Among the divalent counter-ions considered, Ca2+ gave the strongest interaction with the heparin polyanion, but no specific complex formation was observed. O.r.d. and c.d. data are discussed on the basis of a randomly coiled structure for macromolecules composed of rigid, heterocyclic repeating-units that are independent of each other in so far as electronic transitions of chromophore groups contributing to optical activity are concerned. 相似文献
15.
Pore properties of rat brain II sodium channels mutated in the selectivity filter domain 总被引:6,自引:0,他引:6
Thomas Schlief Roland Schönherr Keiji Imoto S. H. Heinemann 《European biophysics journal : EBJ》1996,25(2):75-91
Ion selectivity of voltage-activated sodium channels is determined by amino-acid residues in the pore regions of all four
homologous repeats. The major determinants are the residues DEKA (for repeats I-IV) which form a putative ring structure in
the pore; the homologous structure in Ca-channels consists of EEEE. By combining site-directed mutagenesis of a non-inactivating
form of the rat brain sodium channel II with electrophysiological methods, we attempted to quantify the importance of charge,
size, and side-chain position of the amino-acid residues within this ring structure on channel properties such as monovalent
cation selectivity, single-channel conductance, permeation and selectivity of divalent cations, and channel block by extracellular
Ca2+ and tetrodotoxin (TTX). In all mutant channels tested, even those with the same net charge in the ring structure as the wild
type, the selectivity for Na+ and Li+ over K+, Rb+, Cs+, and NH4
+ was significantly reduced. The changes in charge did not correlate in a simple fashion with the single-channel conductances.
Permeation of divalent ions (Ca2+, Ba2+, Sr2+, Mg2+, Mn2+) was introduced by some of the mutations. The IC50 values for the Ca2+ block of Na+ currents decreased exponentially with increasing net negative charge of the selectivity ring. The sensitivity towards channel
block by TTX was reduced in all investigated mutants. Mutations in repeat IV are an exception as they caused smaller effects
on all investigated channel properties compared with the other repeats.
Received: 24 July 1996 / Accepted: 12 September 1996 相似文献
16.
Characterisation of cation binding and gelation of polyuronates by circular dichroism 总被引:2,自引:0,他引:2
The cation-induced gelation of alginates and pectins with various metal ions has been monitored by circular dichroism (c.d.), using a controlled diffusion technique to prepare homogeneous gels in situ. Spectral changes observed with Ca2+ are closely similar to those previously reported for Ca2+-induced dimerisation of alginate poly-l-guluronate and pectin poly-d-galacturonate chain-sequences in solution, and the magnitude of the c.d. change on gel formation is directly related to the proportion of these structural types present. It therefore appears that gel formation does not introduce optical artefacts such as have been reported for particulate systems or biological membranes. Similar spectral changes are observed on gelation of pectin with Sr2+, Ba2+, Cd2+, Ni2+, or Pb2+, but with minor alterations in the wavelength of maximum c.d. change. These subtle differences are interpreted as reflecting variation in binding-site geometry to accommodate ions of different size. Differences in c.d. behaviour with Mg2+, Ca2+, and Sr2+ are far greater for alginate than for pectin, consistent with the greater selectivity of ion-binding. Gelation of both alginate and pectin with Cu2+ is accompanied by spectral changes that are opposite in sign to those observed with other divalent cations, and span a much wider range of wavelengths. This suggests a different and less-specific binding mechanism, consistent with the known lack of selectivity of Cu2+ for different polyuronates. However, for alginate, there is also evidence of some specific interchain chelation. A minor enhancement of alginate c.d. in the presence of K+ ions is attributed to a decrease in charge density of the polymer chain by bound cations, with consequent increase in segment-segment association in solution. The sign and magnitude of this effect confirm the selectivity of polyuronates for divalent cation. 相似文献
17.
Hana Votavová Dana Kucerová Jürgen Felsberg Jaroslav Sponar 《Journal of biomolecular structure & dynamics》2013,31(3):477-489
Abstract Circular dichroism spectroscopy, absorption spectroscopy, measurements of Tm values, sedimentation analysis and electron microscopy were used to study properties of calf thymus DNA in methanol-water mixtures as a function of monovalent cation (Na+ or Cs+) concentration and also in the presence of divalent cations Ca2+, Mg2+, and Mn2+. In the absence of divalent cations only slight conformational changes occured and no condensation and/or aggregation could be detected. The Tm values depend on the amount of methanol and on the nature and concentration of cations. In methanol-water mixtures higher thermal stability was observed in solutions containing Cs+ ions. Up to 40% (v/v) methanol the addition of divalent ions leads to DNA stabilization. At methanol concentration higher than 50% the presence of divalent cations causes DNA condensation and denaturation even at room temperature. The denaturation is reversible with respect to EDTA addition indicating that no separation of complementary strands occured and the resulting form of DNA is probably similar to the P form. DNA destacking appears to be a direct consequence of stronger cation binding by the condensed DNA in methanol-water mixtures. 相似文献
18.
A comparative study on intrinsic fluorescence of BSA and lysozyme proteins in presence of different divalent ions from their solution and thin film conformations
下载免费PDF全文
![点击此处可从《Luminescence》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Optical emission behaviours of lysozyme and bovine serum albumin, from bulk and thin film geometry, were studied in the presence of three different divalent ions (Mg2+, Ca2+ or Ba2+) using different spectroscopic [steady‐state fluorescence, UV–Vis and Fourier transform infra‐red (FTIR)] techniques. Additionally, protein thin films on silicon surfaces were prepared and morphological studies were carried out using atomic force microscopy. Dynamic quenching was mainly identified for both proteins in the presence of Mg2+, Ca2+ and Ba2+ ions. The molecular conformation of the proteins was modified in thin films compared with that in solution, consequently quenching efficiencies also varied. ATR‐FTIR studies confirmed the conformational changes of proteins in the presence of all divalent ions. All metal ions used were divalent in nature and belonged to the same group of the periodic table but, depending on their individual characteristics such as electron affinity, ionic radius, etc., the magnitude of the protein and hydrated ion interaction varied and accordingly the quenching efficiency was modified. Quenching was maximum for Ca2+ ions, followed by the other two ions. Our study clearly illustrates the geometry‐dependent physical and biological functions of proteins. 相似文献
19.
Xiaozhen Yu Marie-Paule Strub Travis J. Barnard Nicholas Noinaj Grzegorz Piszczek Susan K. Buchanan Justin W. Taraska 《PloS one》2014,9(4)
Many fluorescent proteins have been created to act as genetically encoded biosensors. With these sensors, changes in fluorescence report on chemical states in living cells. Transition metal ions such as copper, nickel, and zinc are crucial in many physiological and pathophysiological pathways. Here, we engineered a spectral series of optimized transition metal ion-binding fluorescent proteins that respond to metals with large changes in fluorescence intensity. These proteins can act as metal biosensors or imaging probes whose fluorescence can be tuned by metals. Each protein is uniquely modulated by four different metals (Cu2+, Ni2+, Co2+, and Zn2+). Crystallography revealed the geometry and location of metal binding to the engineered sites. When attached to the extracellular terminal of a membrane protein VAMP2, dimeric pairs of the sensors could be used in cells as ratiometric probes for transition metal ions. Thus, these engineered fluorescent proteins act as sensitive transition metal ion-responsive genetically encoded probes that span the visible spectrum. 相似文献
20.
《Biophysical journal》2019,116(12):2400-2410
The highly charged RNA molecules, with each phosphate carrying a single negative charge, cannot fold into well-defined architectures with tertiary interactions in the absence of ions. For ribozymes, divalent cations are known to be more efficient than monovalent ions in driving them to a compact state, although Mg2+ ions are needed for catalytic activities. Therefore, how ions interact with RNA is relevant in understanding RNA folding. It is often thought that most of the ions are territorially and nonspecifically bound to the RNA, as predicted by the counterion condensation theory. Here, we show using simulations of Azoarcus ribozyme, based on an accurate coarse-grained three-site interaction model with explicit divalent and monovalent cations, that ion condensation is highly specific and depends on the nucleotide position. The regions with high coordination between the phosphate groups and the divalent cations are discernible even at very low Mg2+ concentrations when the ribozyme does not form tertiary interactions. Surprisingly, these regions also contain the secondary structural elements that nucleate subsequently in the self-assembly of RNA, implying that ion condensation is determined by the architecture of the folded state. These results are in sharp contrast to interactions of ions (monovalent and divalent) with rigid charged rods, in which ion condensation is uniform and position independent. The differences are explained in terms of the dramatic nonmonotonic shape fluctuations in the ribozyme as it folds with increasing Mg2+ or Ca2+ concentration. 相似文献