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1.
The relationship between formation of active in-line attack conformations and monovalent (Na+) and divalent (Mg2+) metal ion binding in hammerhead ribozyme (HHR) has been explored with molecular dynamics simulations. To stabilize repulsions between negatively charged groups, different requirements of the threshold occupancy of metal ions were observed in the reactant and activated precursor states both in the presence and in the absence of a Mg2+ in the active site. Specific bridging coordination patterns of the ions are correlated with the formation of active in-line attack conformations and can be accommodated in both cases. Furthermore, simulation results suggest that the HHR folds to form an electronegative recruiting pocket that attracts high local concentrations of positive charge. The present simulations help to reconcile experiments that probe the metal ion sensitivity of HHR catalysis and support the supposition that Mg2+, in addition to stabilizing active conformations, plays a specific chemical role in catalysis.  相似文献   

2.
Summary (1) When salts are added to buffered suspensions of membrane fragments containing the fluorochrome 1-anilino-8-naphthalenesulfonate (ANS), there is an increased fluorescence. This is caused by increased binding of the fluorochrome; the intrinsic fluorescence characteristics of the bound dye remain unaltered. These properties make ANS a sensitive and versatile indicator of ion association equilibria with membranes. (2) Alkali metal and alkylammonium cations bind to membranes in a unique manner. Cs+ binds most strongly to rat brain microsomal material, with the other alkali metals in the order Cs+>Rb+>K+>Na+>Li+. The reaction is endothermic and entropy driven. Monovalent cations are displaced by other monovalent cations. Divalent cations and some drugs (e. g., cocaine) displace monovalent cations more strongly. (3) Divalent cations bind to membranes (and to lecithin micelles) at four distinct sites, having apparent association constants between 50 and 0.2mm –1. The characteristics of the titration suggest that only one species of binding site is present at any one time, and open the possibility that structural transitions of the unassociated coordination sites may be induced by divalent cation binding. Divalent cation binding at the weakest site (like monovalent cation binding) is endothermic and entropy driven. At the next stronger site, the reaction is exothermic. Monovalent cations affect divalent cation binding by reducing the activity coefficient: they do not appear to displace divalent cations from their binding sites.  相似文献   

3.
The hammerhead ribozyme is generally accepted as a well characterized metalloenzyme. However, the precise nature of the interactions of the RNA with metal ions remains to be fully defined. Examination of metal ion-catalyzed hammerhead reactions at limited concentrations of metal ions is useful for evaluation of the role of metal ions, as demonstrated in this study. At concentrations of Mn2+ ions from 0.3 to 3 mM, addition of the ribozyme to the reaction mixture under single-turnover conditions enhances the reaction with the product reaching a fixed maximum level. Further addition of the ribozyme inhibits the reaction, demonstrating that a certain number of divalent metal ions is required for proper folding and also for catalysis. At extremely high concentrations, monovalent ions, such as Na+ ions, can also serve as cofactors in hammerhead ribozyme-catalyzed reactions. However, the catalytic efficiency of monovalent ions is extremely low and, thus, high concentrations are required. Furthermore, addition of monovalent ions to divalent metal ion-catalyzed hammerhead reactions inhibits the divalent metal ion-catalyzed reactions, suggesting that the more desirable divalent metal ion–ribozyme complexes are converted to less desirable monovalent metal ion–ribozyme complexes via removal of divalent metal ions, which serve as a structural support in the ribozyme complex. Even though two channels appear to exist, namely an efficient divalent metal ion-catalyzed channel and an inefficient monovalent metal ion-catalyzed channel, it is clear that, under physiological conditions, hammerhead ribozymes are metalloenzymes that act via the significantly more efficient divalent metal ion-dependent channel. Moreover, the observed kinetic data are consistent with Lilley’s and DeRose’s two-phase folding model that was based on ground state structure analyses.  相似文献   

4.
Summary A nonselective cation channel activated by patch excision was characterized in inside-out patches from spiny lobster olfactory receptor neurons. The channel, which was permeable to Na+, K+ and Cs+, had a conductance of 320 pS and was weakly voltage dependent in the presence of micromolar divalent cations. Millimolar internal divalent cations caused a voltage-and concentration-dependent block of Na+ permeation. Analysis of the voltage dependence indicated that the proportion of the membrane's electric field sensed by Mg2+ was >1, suggesting that the channel contains a multi-ion pore. Internal divalent cations also reduced the frequency of channel opening in a concentration-dependent, but not voltage-dependent, manner, indicating that different cation binding sites affect gating and conductance. While block of gating prevented determining if internal divalent cations permeate the channel, a channel highly permeable to external divalent cations was observed upon patch excision to the inside-out configuration. The monovalent and divalent cation conductances shared activation by patch excision, weak voltage dependence, and steady-state activity, suggesting that they are the same channel. These data extend our understanding of this type of channel by demonstrating permeation by monovalent cations, detailing Mg2+ block of Na permeation, and demonstrating the channel's presence in arthropods.  相似文献   

5.
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   

6.
《Biophysical journal》2020,118(4):909-921
In the era of opioid abuse epidemics, there is an increased demand for understanding how opioid receptors can be allosterically modulated to guide the development of more effective and safer opioid therapies. Among the modulators of the μ-opioid (MOP) receptor, which is the pharmacological target for the majority of clinically used opioid drugs, are monovalent and divalent cations. Specifically, the monovalent sodium cation (Na+) has been known for decades to affect MOP receptor signaling by reducing agonist binding, whereas the divalent magnesium cation (Mg2+) has been shown to have the opposite effect, notwithstanding the presence of sodium chloride. Although ultra-high-resolution opioid receptor crystal structures have revealed a specific Na+ binding site and molecular dynamics (MD) simulation studies have supported the idea that this monovalent ion reduces agonist binding by stabilizing the receptor inactive state, the putative binding site of Mg2+ on the MOP receptor, as well as the molecular determinants responsible for its positive allosteric modulation of the receptor, are unknown. In this work, we carried out tens of microseconds of all-atom MD simulations to investigate the simultaneous binding of Mg2+ and Na+ cations to inactive and active crystal structures of the MOP receptor embedded in an explicit lipid-water environment and confirmed adequate sampling of Mg2+ ion binding with a grand canonical Monte Carlo MD method. Analyses of these simulations shed light on 1) the preferred binding sites of Mg2+ on the MOP receptor, 2) details of the competition between Mg2+ and Na+ cations for specific sites, 3) estimates of binding affinities, and 4) testable hypotheses of the molecular mechanism underlying the positive allosteric modulation of the MOP receptor by the Mg2+ cation.  相似文献   

7.
The hammerhead cleavage reaction in monovalent cations   总被引:10,自引:3,他引:7       下载免费PDF全文
Recently, Murray et al. (Chem Biol, 1998, 5:587-595) found that the hammerhead ribozyme does not require divalent metal ions for activity if incubated in high (> or =1 M) concentrations of monovalent ions. We further characterized the hammerhead cleavage reaction in the absence of divalent metal. The hammerhead is active in a wide range of monovalent ions, and the rate enhancement in 4 M Li+ is only 20-fold less than that in 10 mM Mg2+. Among the Group I monovalent metals, rate correlates in a log-linear manner with ionic radius. The pH dependence of the reaction is similar in 10 mM Mg2+, 4 M Li+, and 4 M Na+. The exchange-inert metal complex Co(NH3)3+ also supports substantial hammerhead activity. These results suggest that a metal ion does not act as a base in the reaction, and that the effects of different metal ions on hammerhead cleavage rates primarily reflect structural contributions to catalysis.  相似文献   

8.
In the E1 state of the Na,K-ATPase all cations present in the cytoplasm compete for the ion binding sites. The mutual effects of mono-, di- and trivalent cations were investigated by experiments with the electrochromic fluorescent dye RH421. Three sites with significantly different properties could be identified. The most unspecific binding site is able to bind all cations, independent of their valence and size. The large organic cation Br2-Titu3+ is bound with the highest affinity (<μm), among the tested divalent cations Ca2+ binds the strongest, and Na+ binds with about the same equilibrium dissociation constant as Mg2+ (∼0.8 mm). For alkali ions it exhibits binding affinities following the order of Rb+≃ K+ > Na+ > Cs+ > Li+. The second type of binding site is specific for monovalent cations, its binding affinity is higher than that of the first type, for Na+ ions the equilibrium dissociation constant is < 0.01 mm. Since binding to that site is not electrogenic it has to be close to the cytoplasmic surface. The third site is specific for Na+, no other ions were found to bind, the binding is electrogenic and the equilibrium dissociation constant is 0.2 mm. Received: 7 August 2000/Revised: 14 November 2000  相似文献   

9.
We have characterized a novel Zn2+-catalyzed cleavage site between nucleotides C3 and U4 in the catalytic core of the hammerhead ribozyme. In contrast to previously described divalent metal-ion-dependent cleavage of RNA, U4 cleavage is only observed in the presence of Zn2+. This new cleavage site has an unusual pH dependence, in that U4 cleavage products are only observed above pH 7.9 and reach a maximum yield at about pH 8.5. These data, together with the fact that no metal ion-binding site is observed in proximity to the U4 cleavage site in either of the crystal structures, point toward a pH-dependent conformational change in the hammerhead ribozyme. We have described previously Zn2+-dependent cleavage between G8 and A9 in the hammerhead ribozyme and have discovered that U4 cleavage occurs only after A9 cleavage. To our knowledge, this is the first example of sequential cleavage events as a possible regulatory mechanism in ribozymes.  相似文献   

10.
A theory is presented on the electrostatic properties of the surface area of phosphatidyl-glycerol monolayers spreading at an air-water interface in the presence of monovalent and divalent cations. In the present theory, the adsorption of monovalent and divalent cations to the membranes is taken into account, besides the dissociation of protons, as a possible cause of the change of surface charge density with the variation of pH or ion concentrations. It is also pointed out that, in the presence of structure-making ions such as Li+ and Na+, the nearest-neighbour interactions between proton dissociation sites become important for the monolayers in the gel state to yield a sharp expansion of the surface area with the increase of pH. The present theory shows quantitative agreements with previously-observed data.  相似文献   

11.
Bacteriorhodopsin (bR) is the prototype of an integral membrane protein with seven membrane-spanning α-helices and serves as a model of the G-protein-coupled drug receptors. This study is aimed at reaching a greater understanding of the role of amine local anesthetic cations on the proton transport in the bR protein, and furthermore, the functional role of “the cation” in the proton pumping mechanism. The effect of the amine anesthetic cations on the proton pump in the bR blue membrane was compared with those by divalent (Ca2+, Mg2+ and Mn2+) and monovalent metal cations (Li+, Na+, K+ and Cs+), which are essential for the correct functioning of the proton pumping of the bR protein. The results suggest that the interacting site of the divalent cation to the bR membrane may differ from that of the monovalent metal cation. The electric current profile of the bR blue membrane in the presence of the amine anesthetic cations was biphasic, involving the generation and inhibition of the proton pumping activity in a concentration-dependent manner. The extent of the regeneration of the proton pump by the additives increased in the order of monovalent metal cation<monovalent amine anesthetic cation<divalent metal cation. We found that organic cations such as the amine anesthetics can also regenerate the proton pump in the bR protein. The inhibition of proton transport in the bR protein by the anesthetic cations was elucidated using the wild type, the E204Q and the D96N mutated bRs. The hydrophobic interaction of the amine anesthetics with the bR protein plays an important part in inhibiting the bR proton pump.  相似文献   

12.
《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   

13.
Nucleic acids generally reside in cellular aqueous solutions with mixed divalent/monovalent ions, and the competitive binding of divalent and monovalent ions is critical to the structures of nucleic acids because of their polyanionic nature. In this work, we first proposed a general and effective method for simulating a nucleic acid in mixed divalent/monovalent ion solutions with desired bulk ion concentrations via molecular dynamics (MD) simulations and investigated the competitive binding of Mg2+/Na+ ions to various nucleic acids by all-atom MD simulations. The extensive MD-based examinations show that single MD simulations conducted using the proposed method can yield desired bulk divalent/monovalent ion concentrations for various nucleic acids, including RNA tertiary structures. Our comprehensive analyses show that the global binding of Mg2+/Na+ to a nucleic acid is mainly dependent on its structure compactness, as well as Mg2+/Na+ concentrations, rather than the specific structure of the nucleic acid. Specifically, the relative global binding of Mg2+ over Na+ is stronger for a nucleic acid with higher effective surface charge density and higher relative Mg2+/Na+ concentrations. Furthermore, the local binding of Mg2+/Na+ to a phosphate of a nucleic acid mainly depends on the local phosphate density in addition to Mg2+/Na+ concentrations.  相似文献   

14.
Although the hammerhead reaction proceeds most efficiently in divalent cations, cleavage in 4 M LiCl is only approximately 10-fold slower than under standard conditions of 10 mM MgCl2 (Murray et al., Chem Biol, 1998, 5:587-595; Curtis & Bartel, RNA, 2001, this issue, pp. 546-552). To determine if the catalytic mechanism with high concentrations of monovalent cations is similar to that with divalent cations, we compared the activities of a series of modified hammerhead ribozymes in the two ionic conditions. Nearly all of the modifications have similar deleterious effects under both reaction conditions, suggesting that the hammerhead adopts the same general catalytic structure with both monovalent and divalent cations. However, modification of three ligands previously implicated in the binding of a functional divalent metal ion have substantially smaller effects on the cleavage rate in Li+ than in Mg2+. This result suggests that an interaction analogous to the interaction made by this divalent metal ion is absent in the monovalent reaction. Although the contribution of this divalent metal ion to the overall reaction rate is relatively modest, its presence is needed to achieve the full catalytic rate. The role of this ion appears to be in facilitating formation of the active structure, and any direct chemical role of metal ions in hammerhead catalysis is small.  相似文献   

15.
Isolated liver nuclei were extracted 3 times at pH 7.2 with solutions containing either (1) monovalent cations, (2) both mono- and divalent cations, or (3) sucrose solutions containing only divalent cations. The extracted proteins were analysed by two-dimensional acrylamide gel electrophoresis and the ultrastructural alterations of the treated nuclei were examined by electron microscopy. The solutions containing Na+ or K+ monovalent and Ca2+ and Mg2+ divalent ions extracted the same amount (18–22 %) of the nuclear proteins. The two-dimensional gel electrophoretic patterns of these extracts were nearly identical and the structures of the nuclear components were well preserved even after 3 times repeated extractions. The solution containing only Na+ extracted less protein (14–15 %) than the solutions containing both mono- and divalent cations. Extraction with isotonic NaCl solution altered the nuclear and nucleolar morphology; unlike the other solutions employed, this solution extracted some DNA and histones. The isotonic sucrose solution containing only divalent cations extracted less protein than the other solutions (9–11 %) and produced marked condensation of the chromatin. These analytical and electron microscopic studies showed that mono- and divalent cations play a role in structural organization of chromatin.  相似文献   

16.
The yeast Debaryomyces hansenii is considered a marine organism. Sea water contains 0.6 M Na+ and 10 mM K+; these cations permeate into the cytoplasm of D. hansenii where proteins and organelles have to adapt to high salt concentrations. The effect of high concentrations of monovalent and divalent cations on isolated mitochondria from D. hansenii was explored. As in S. cerevisiae, these mitochondria underwent a phosphate-sensitive permeability transition (PT) which was inhibited by Ca2+ or Mg2+. However, D. hansenii mitochondria require higher phosphate concentrations to inhibit PT. In regard to K+ and Na+, and at variance with mitochondria from all other sources known, these monovalent cations promoted closure of the putative mitochondrial unspecific channel. This was evidenced by the K+/Na+-promoted increase in: respiratory control, transmembrane potential and synthesis of ATP. PT was equally sensitive to either Na+ or K+. In the presence of propyl-gallate PT was still observed while in the presence of cyanide the alternative pathway was not active enough to generate a ΔΨ due to a low AOX activity. In D. hansenii mitochondria K+ and Na+ optimize oxidative phosphorylation, providing an explanation for the higher growth efficiency in saline environments exhibited by this yeast.  相似文献   

17.
Golden BL 《Biochemistry》2011,50(44):9424-9433
The hepatitis delta virus (HDV) ribozyme and related RNAs are widely dispersed in nature. This RNA is a small nucleolytic ribozyme that self-cleaves to generate products with a 2',3'-cyclic phosphate and a free 5'-hydroxyl. Although small ribozymes are dependent on divalent metal ions under biologically relevant buffer conditions, they function in the absence of divalent metal ions at high ionic strengths. This characteristic suggests that a functional group within the covalent structure of small ribozymes is facilitating catalysis. Structural and mechanistic analyses have demonstrated that the HDV ribozyme active site contains a cytosine with a perturbed pK(a) that serves as a general acid to protonate the leaving group. The reaction of the HDV ribozyme in monovalent cations alone never approaches the velocity of the Mg(2+)-dependent reaction, and there is significant biochemical evidence that a Mg(2+) ion participates directly in catalysis. A recent crystal structure of the HDV ribozyme revealed that there is a metal binding pocket in the HDV ribozyme active site. Modeling of the cleavage site into the structure suggested that this metal ion can interact directly with the scissile phosphate and the nucleophile. In this manner, the Mg(2+) ion can serve as a Lewis acid, facilitating deprotonation of the nucleophile and stabilizing the conformation of the cleavage site for in-line attack of the nucleophile at the scissile phosphate. This catalytic strategy had previously been observed only in much larger ribozymes. Thus, in contrast to most large and small ribozymes, the HDV ribozyme uses two distinct catalytic strategies in its cleavage reaction.  相似文献   

18.
The cation discriminations of salinomycin and its derivatives have been studied by measuring complexability with cations and transport rate of them across organic phase. Salinomycin exhibited a great preference for K+ over other monovalent and divalent cations in migrating cations into organic phase in two phase systems. The antibiotic mediated the transport of Na+ and Rb+ as effectively as that of K+ across CCl4 bulk phase, but not those of Cs+, Mg2+, Ca2+, Sr2+. From the above results, salinomycin is concluded to act as an alkali ion carrier. The OH-acylated salinomycins retained the activity of parent compound, but the COOH-esterified salinomycins lost the activity.  相似文献   

19.
Several observations have already suggested that the carboxyl groups are involved in the association of divalent cations with bacteriorhodopsin (Chang et al., 1985). Here we show that at least part of the protons released from deionized purple membrane (`blue membrane') samples when salt is added are from carboxyl groups. We find that the apparent pK of magnesium binding to purple membrane in the presence of 0.5 mM buffer is 5.85. We suggest this is the pK of the carboxyl groups shifted from their usual pK because of the proton concentrating effect of the large negative surface potential of the purple membrane. Divalent cations may interact with negatively charged sites on the surface of purple membrane through the surface potential and/or through binding either by individual ligands or by conformation-dependent chelation. We find that divalent cations can be released from purple membrane by raising the temperature. Moreover, purple membrane binds only about half as many divalent cations after bleaching. Neither of these operations is expected to decrease the surface potential and thus these experiments suggest that some specific conformation in purple membrane is essential for the binding of a substantial fraction of the divalent cations. Divalent cations in purple membrane can be replaced by monovalent, (Na+ and K+), or trivalent, (La+++) cations. Flash photolysis measurements show that the amplitude of the photointermediate, O, is affected by the replacement of the divalent cations by other ions, especially by La+++. The kinetics of the M photointermediate and light-induced H+ uptake are not affected by Na+ and K+, but they are drastically lengthened by La+++ substitution, especially at alkaline pHs. We suggest that the surface charge density and thus the surface potential is controlled by divalent cation binding. Removal of the cations (to make deionized blue membrane) or replacement of them (e.g. La+++-purple membrane) changes the surface potential and hence the proton concentration near the membrane surface. An increase in local proton concentration could cause the protonation of critical carboxyl groups, for example the counter-ion to the protonated Schiff's base, causing the red shift associated with the formation of both deionized and acid blue membrane. Similar explanations based on regulation of the surface proton concentration can explain many other effects associated with the association of different cations with bacteriorhodopsin.  相似文献   

20.
Renilla lumisomes produce a bioluminescent flash when the vesicles are disrupted with hypotonic solutions containing Ca2+. A flash is also observed in the presence of Ca2+ using isotonic solutions of monovalent cations under the following conditions: When the Na+K+ ratio inside the lumisomal membrane is high and when this ratio outside the membrane is low. We suggest that Na+ may be the counter ion for Ca2+ transport. Na+, when outside the membrane, inhibits Ca2+-triggered luminescence suggesting that Na+ blocks Ca2+ channels. Ca2+ uptake into the lumisomal membrane, as measured by bioluminescence, is very rapid in the presence of the ionophore A23187. X537A is much less effective. The Ca2+ triggered bioluminescence flash observed with lumisomes provides a rapid and sensitive assay for ionophores that are specific for divalent cations such as Ca2+.  相似文献   

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