Yeast enolase carboxyl modification using Woodward's reagent K

Yeast enolase is inactivated by Woodward's reagent K. Substantial protection is afforded by binding of 1 mol of "conformational" metal ion/subunit. Inactivation is correlated with modification of 13 carboxyl groups/subunit in the absence of conformational metal ion and 17 in its presence. Ten tryptic peptides labeled by Woodward's reagent K can be isolated, mostly from the C-terminal half of the protein. The changes in reactivity of these peptides produced by conformational metal ion suggest direct coordination to Glu-181 together with a contraction of the protein.     

Studies of the role of catalytic and conformational metals in producing enzymatic activity in yeast enolase

Spectrophotometric titrations of yeast apoenolase with magnesium, the metal that produces the highest level of activity, nickel, which produces a very low level, and calcium, which produces no activity, suggest strong binding of 2 mol (1 per subunit) of all three metals at the same sites, called “conformational” sites. About two-thirds of the possible absorbance change in the chromophoric competitive inhibitor 3-aminoenolpyruvate-2-phosphate (AEP) that occurs when it binds to the enzyme in the presence of saturating levels of magnesium is produced when just 2 mol (1 per subunit) of magnesium is added. Since additional “catalytic” metal won't bind unless the AEP does, and the AEP won't bind unless the “conformational” sites are filled with metal, much of the absorbance change in the AEP must be produced by conformational metal.Metals that do not produce enzymatic activity do not produce the absorbance change in AEP whereas metals that permit any level of enzymatic activity produce the same absorbance change that magnesium does-the reaction is “all or none.” Studies of the effect of calcium, nickel, and magnesium on the CD spectrum of apoenzyme-AEP solutions suggest that activating metals produce an asymmetric chromophore in the AEP. This is interprested as indicating the chromophore in AEP bound to enzyme in the presence of an activating metal is a twisted carbon-carbon double bond.Calorimetric studies show the competitive inhibitor 3-phosphoglycerate binds to the calcium- and magnesium-enzyme with about the same change in enthalpy. The substrate or AEP reduces the rate of the apparent reaction of the calcium- or magnesium-enzyme with excess EDTA, suggesting that both substrate and AEP bind to the calcium-enzyme. The interpretation of these data is that the conformational metal plays a crucial role in activating the substrate while the catalytic metal controls the reaction rate. This interpretation is supported by experiments in which an enzyme with one type of conformational metal is reacted in the stopped-flow with catalytic metal and substrate. If an activating metal is the conformational metal, the initial activity is greater.     

A Usage Scenario Independent “Air Chargeable” Flexible Zinc Ion Energy Storage Device

A rationally designed “air chargeable” energy storage device is demonstrated, which can be effectively charged by harvesting pervasive energy from the ambient environment. For an “air chargeable” zinc‐ion capacitor system, the system simply consists of a flexible bifunctional “U” shaped electrode (with the functions of energy harvesting and storage), a zinc metal electrode in middle, and two different polyelectrolytes (polyacrylamide and sodium polyacrylate) sandwiched between the zinc metal and “U” shaped electrode. When the zinc‐ion capacitor is exhausted, it can be quickly charged to 88% within 10 min by simply opening the sealing tape and allowing the air diffuse in. The capacitor exhausting‐air charging processes are repeated 60 times and the whole system works well. When the external power supplier is available, both the zinc‐ion capacitor and “air charging” component can be fully recovered. A large capacity (≈1000 mAh) “air chargeable” zinc‐vanadium battery is also demonstrated. The zinc‐vanadium battery can be fully charged by air in 1 h. This work offers a usage scenario independent reliable self‐chargeable power supply system as a promising approach to solve the intermittent and unpredictable nature of currently developed self‐chargeable devices.     

Model peptide‐based system used for the investigation of metal ions binding to histidine‐containing polypeptides

The reaction of histidine‐containing polypeptides with toxic and essential metals and the molecular mechanism of complexation has yet to be determined, particularly with respect to the conformational changes of the interacting macromolecules. Therefore, a system of oligopeptides containing histidine residues in various positions of Ala or Gly sequences has been designed and used in heavy metal comparatively binding experiments. The role of spacing residues (Gly and Ala repeats) in selecting the various conformations was investigated. The newly synthesized peptides and metal ion adducts have been characterized by Fourier transform infrared spectroscopy (FTIR) as well as electrospray ion trap mass spectrometry (ESI–MS) and circular dichroism (CD). The analysis of CD‐spectra of the four peptides in water revealed that the secondary structure depends much on the position of each amino acid in the peptide backbone. Our peptides system reveals various binding mechanisms of metal ions to peptides depending on the position of histidine residue and the corresponding conformations of Ala or Gly sequences. Biological and medical consequences of conformational changes of metal‐bound peptides are further discussed. Thus, the binding of heavy metals to four peptides may serve as a model system with respect to the conformational consequences of the metal addition on the amino acid repeats situated in prion protein. © 2010 Wiley Periodicals, Inc. Biopolymers 93:497–508, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Yeast Enolase: Mechanism of Activation by Metal Ion

Abstract

The significance of the “conformational-catalytic” mechanism of metal ion activation is not known. It is still at the state of being established for some enzymes. Right now, only speculations can be offered. All the enzymes mentioned have subunits and all have strongly negatively charged phosphate-containing substrates.

As a preliminary hypothesis, it may be suggested that one of the 2 mol of metal ion involved functions to “lock” one of the substrates into a configuration so that when the second one attacks that or another substrate, electrons are withdrawn from crucial bonds and catalysis occurs. Either metal ion or both or neither may do this by interacting directly with a substrate.     

Side by Side Battery Technologies with Lithium‐Ion Based Batteries

In recent years, the electrochemical power sources community has launched massive research programs, conferences, and workshops on the “post Li battery era.” However, in this report it is shown that the quest for post Li‐ion and Li battery technologies is incorrect in its essence. This is the outcome of a three day discussion on the future technologies that could provide an answer to a question that many ask these days: Which are the technologies that can be regarded as alternative to Li‐ion batteries? The answer to this question is a rather surprising one: Li‐ion battery technology will be here for many years to come, and therefore the use of “post Li‐ion” battery technologies would be misleading. However, there are applications with needs for which Li‐ion batteries will not be able to provide complete technological solutions, as well as lower cost and sustainability. In these specific cases, other battery technologies will play a key role. Here, the term “side‐by‐side technologies” is coined alongside a discussion of its meaning. The progress report does not cover the topic of Li‐metal battery technologies, but covers the technologies of sodium‐ion, multivalent, metal–air, and flow batteries.     

DNA Topoisomerase Inhibitors: Trapping a DNA-Cleaving Machine in Motion

Type II topoisomerases regulate DNA topology by making a double-stranded break in one DNA duplex, transporting another DNA segment through this break and then resealing it. Bacterial type IIA topoisomerase inhibitors, such as fluoroquinolones and novel bacterial topoisomerase inhibitors, can trap DNA cleavage complexes with double- or single-stranded cleaved DNA. To study the mode of action of such compounds, 21 crystal structures of a “gyrase^CORE” fusion truncate of Staphyloccocus aureus DNA gyrase complexed with DNA and diverse inhibitors have been published, as well as 4 structures lacking inhibitors. These structures have the DNA in various cleavage states and appear to track trajectories along the catalytic paths of the DNA cleavage/religation steps. The various conformations sampled by these multiple “gyrase^CORE” structures show rigid body movements of the catalytic GyrA WHD and GyrB TOPRIM domains across the dimer interface. Conformational changes common to all compound-bound structures suggest common mechanisms for DNA cleavage-stabilizing compounds. The structures suggest that S. aureus gyrase uses a single moving-metal ion for cleavage and that the central four base pairs need to be stretched between the two catalytic sites, in order for a scissile phosphate to attract a metal ion to the A-site to catalyze cleavage, after which it is “stored” in another coordination configuration (B-site) in the vicinity. We present a simplified model for the catalytic cycle in which capture of the transported DNA segment causes conformational changes in the ATPase domain that push the DNA gate open, resulting in stretching and cleaving the gate-DNA in two steps.     

Crystal structure of the coxsackievirus A16 RNA-dependent RNA polymerase elongation complex reveals novel features in motif A dynamics

<正>Dear Editor,Coxsackievirus A16(CV A16)and enterovirus 71(EV71)are currently the two primary causative agents of handfoot-and-mouth disease(HFMD)(Solomon et al.,2010;Mao et al.,2014),threatening health of children worldwide.They both belong to the Enterovirus genus of the     

Partial Metal Ion Saturation of C2 Domains Primes Synaptotagmin 1-Membrane Interactions

Synaptotagmin 1 (Syt1) is an integral membrane protein whose phospholipid-binding tandem C2 domains, C2A and C2B, act as Ca²⁺ sensors of neurotransmitter release. Our objective was to understand the role of individual metal-ion binding sites of these domains in the membrane association process. We used Pb²⁺, a structural and functional surrogate of Ca²⁺, to generate the protein states with well-defined protein-metal ion stoichiometry. NMR experiments revealed that binding of one divalent metal ion per C2 domain results in loss of conformational plasticity of the loop regions, potentially pre-organizing them for additional metal-ion and membrane-binding events. In C2A, a divalent metal ion in site 1 is sufficient to drive its weak association with phosphatidylserine-containing membranes, whereas in C2B, it enhances the interactions with the signaling lipid phosphatidylinositol-4,5-bisphosphate. In full-length Syt1, both Pb²⁺-complexed C2 domains associate with phosphatidylserine-containing membranes. Electron paramagnetic resonance experiments show that the extent of membrane insertion correlates with the occupancy of the C2 metal ion sites. Together, our results indicate that upon partial metal ion saturation of the intra-loop region, Syt1 adopts a dynamic, partially membrane-bound state. The properties of this state, such as conformationally restricted loop regions and positioning of C2 domains in close proximity to anionic lipid headgroups, “prime” Syt1 for cooperative binding of a full complement of metal ions and deeper membrane insertion.     

Metal Ion Binding and Conformational Transitions in Concanavalin A: A Structure-Function Study

Abstract

The affinity of the lectin Concanavalin A (Con A) for saccharides, and its requirement for metal ions such as Mn²⁺ and Ca²⁺, have been known for about 50 years. However the relationship between metal ion binding and the saccharide binding activity of Con A has only recently been examined in detail. Brown et al. (Biochemistry 16, 3883 (1977)) showed that Con A exists as a mixture of two conformational states: a “locked” form and an “unlocked” form. The unlocked form of the protein weakly binds metal ions and saccharide, and is the predominate conformation of demetallized Con A (apo-Con A) at equilibrium. The locked form binds two metal ions per monomer with the resulting complex(es) possessing full saccharide binding activity. Brown and coworkers measured the kinetics of the transition of the unlocked form to the fully metallized locked conformation containing Mn²⁺and Ca²⁺. They also demonstrated that Mn²⁺ alone could form a locked ternary complex with Con A, and that rapid removal of the ions resulted in a metastable form of apo-Con A in the locked conformation which slowly (hours at 25°C) reverted back to (predominantly) the unlocked conformation. The ability to form either conformation in the absence or presence of metal ions has thus allowed us to explore the relationship between metal ion binding and conformational transitions in Con A as determinants of the saccharide binding activity of the lectin.

Based on the kinetics of the transition of unlocked apo-Con A to fully metallized locked Con A, and X-ray crystallographic data, it appears that the transition between the two conformations of Con A involves a cis-trans isomerization of an Ala-Asp peptide bond in the backbone of the protein, near one of the two metal ion binding sites. The relatively large activation energy for the transition (～ 22 kcal M^?1) results in relatively slow interconversions between the conformations (from minutes to days), whereas the equilibria with metal ions and saccharide are rapid. Thus, many metastable complexes can be formed and a variety of transition pathways between the two conformations studied.

We have identified and characterized binary, ternary, and quaternary complexes of both conformations of Con A containing Mn²⁺ and saccharide, and have determined both metalion and saccharide dissociation constants for all of them, as well as equilibrium and kinetic values for the conformational transitions between them. The main finding is that saccharide binds very weakly (K_d～2 M) to unlocked apo-Con A and very tightly to the locked ternary Mn²⁺-Con A complex (K_d～ 10^?4 M). Saccharide binding increases along the various pathways connecting these two species in a nonadditive fashion. Thus, both conformation and metal ion binding determine the saccharide affinity of each complex, although the specificity of saccharide binding of the various species is maintained throughout.     

Evidence for methionine sulfoximine as a transition-state analog for glutamine synthetase from NMR and EPR data.

Manganese(II)bound at the “tight” metal ion site of unadenylylated glutamine synthetase (E. coli W) has two rapidly exchanging first coordination sphere water molecules. The solvation number was evaluated from a study of the frequency dependence of $\text{1}\text{pT}{}_{\mathrm{<mtext>1p</mtext>}}$, the paramagnetic contribution to the longitudinal relaxation rate of solvent protons. The number of rapidly exchanging water molecules is reduced to one in the presence of saturating L-glutamate and to ～0.2 when L-methionine SR-sulfoximine (MSOX) is present. MSOX is a linear competitive inhibitor (K_I=3μM) of glutamine synthetase when L-Glu is the substrate. The dissociation constant of MSOX measured by following the 18 fold decrease in $\text{1}\text{pT}{}_{\mathrm{1p}}$ (at 48 MHz) is 30μM and is lowered to ～9μM in the presence of ADP. The high affinity of MSOX for the enzyme suggests that this compound mimicks the “transition-state” for the glutamine synthetase reaction. Further evidence for this postulate is found from the dramatic sharpening of the epr spectrum of enzyme-bound Mn(II) in the presence of MSOX and MSOX plus ADP. The intense change in the epr spectrum arises from reduced solvent accessibility to bound Mn(II) and conformational changes produced by binding MSOX and ADP. The suggestion is made from these data that L-Glu and MSOX bind near or directly to the Mn(II) at the “tight” metal ion site in glutamine synthetase isolated from E. coli W.     

Relaxation studies on complex formation of macrocyclic and open chain antibiotics with monovalent cations

The stability constants for the 1 : 1 complexes of macrocyclic antibiotics (nonactin, monactin, dinactin and trinactin) with Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, NH⁺₄ and for the Na⁺-complexes with the open chain compounds nigericin and monensin in methanol solution have been determined. The relaxation amplitude method was employed to obtain both the equilibrium constants and the enthalpies of reaction. The kinetics were studied with the help of temperature-jump, electric-field pulse and ultrasonic absorption techniques. Although complex formation of the metal ions with the antibiotics involves multidentate ligand chelation, the formation rates are in general very high, i.e. close to the limits imposed for diffusion controlled processes. The data for the macrotetrolides indicate the existence of conformational transition prior to complexation. A sequential substitution or “redressing” mechanism is proposed which is in accord with the high rates of complex formation. The selectivity patterns, as expressed by the equilibrium constants, are similar to those observed for the transport of metal ions across membranes in presence of the antibiotics. Selectivity results from an optimal balance between the strength of metal ion solvation and the stability of the individual metal complex, which in turn is governed by the conformational flexibility of the antibiotics.     

Hybrid and Aqueous Lithium‐Air Batteries

Lithium‐air (Li‐air) batteries have become attractive because of their extremely high theoretical energy density. However, conventional Li‐air cells operating with non‐aqueous electrolytes suffer from poor cycle life and low practical energy density due to the clogging of the porous air cathode by insoluble discharge products, contamination of the organic electrolyte and lithium metal anode by moist air, and decomposition of the electrolyte during cycling. These difficulties may be overcome by adopting a cell configuration that consists of a lithium‐metal anode protected from air by a Li⁺‐ion solid electrolyte and an air electrode in an aqueous catholyte. In this type of configuration, a Li⁺‐ion conducting “buffer” layer between the lithium‐metal anode and the solid electrolyte is often necessary due to the instability of many solid electrolytes in contact with lithium metal. Based on the type of buffer layer, two different battery configurations are possible: “hybrid” Li‐air batteries and “aqueous” Li‐air batteries. The hybrid and aqueous Li‐air batteries utilize the same battery chemistry and face similar challenges that limit the cell performance. Here, an overview of recent developments in hybrid and aqueous Li‐air batteries is provided and the factors that influence their performance and impede their practical applications, followed by future directions are discussed.     

Endplate currents are shortened by octanol: Possible role of membrane lipid

The time constant of the decay phase of miniature endplate currents is shortened in the presence of octanol (mM). A change in the power spectrum of acetylcholine “noise” indicates that octanol shortens the duration of “elementary” conductance changes thought to depend on membrane conformational changes produced by acetylcholine. It is proposed that octanol causes these conformational changes to occur more rapidly by increasing the fluidity of membrane lipid.     

Cation-Mediated Interplay of Loops in Chaperonin-10

Abstract

The ubiquitously occurring chaperonins consist of a large tetradecameric Chaperonin-60, forming a cylindrical assembly, and a smaller heptameric Chaperonin-10. For a functional protein folding cycle, Chaperonin-10 caps the cylindrical Chaperonin-60 from one end forming an asymmetric complex. The oligomeric assembly of Chaperonin-10 is known to be highly plastic in nature. In Mycobacterium tuberculosis, the plasticity has been shown to be modulated by reversible binding of divalent cations. Binding of cations confers rigidity to the metal binding loop, and also promotes stability of the oligomeric structure. We have probed the conformational effects of cation binding on the Chaperonin-10 structure through fluorescence studies and molecular dynamics simulations. Fluorescence studies show that cation binding induces reduced exposure and flexibility of the dome loop. The simulations corroborate these results and further indicate a complex landscape of correlated motions between different parts of the molecule. They also show a fascinating interplay between two distantly spaced loops, the metal binding “dome loop” and the GroEL-binding “mobile loop”, suggesting an important cation-mediated role in the recognition of Chaperonin-60. In the presence of cations the mobile loop appears poised to dock onto the Chaperonin-60 structure. The divalent metal ions may thus act as key elements in the protein folding cycle, and trigger a conformational switch for molecular recognition.     

Acetylcholine receptor-controlled ion flux in electroplax membrane vesicles: A minimal mechanism based on rate measurements in the millisecond to minute time region

The dependence of acetylcholine receptor-controlled transmembrane ion flux on carbamylcholine concentration was measured in the msec time region, using membrane vesicles and a quench flow technique. 4 Measurements were made: (1) transmembrane ion influx, (2) rate of inactivation of the receptor by carbamylcholine, (3) rate of recovery, and (4) ion influx mediated by “inactivated” receptor. The minimal model, based on the measurements, accounts for the time dependence of receptor-controlled ion flux over a 200-fold carbamylcholine concentration range. The maximum flux rate of 84 sec^?1 indicates that we have succeeded in measuring the receptor-controlled processes which give rise to electrical signals in cells.     

Parallel Double Helices of DNA. Conformational Analysis of Regular Helices with the Second Order Symmetry Axis

Abstract

We have performed a conformational analysis of DNA double helices with parallel directed backbone strands connected with the second order symmetry axis being at the same time the helix axis. The calculations were made for homopolymers poly(dA) · poly(dA), poly(dC) · poly(dC), poly(dG) poly(dG), and poly(dT) · poly(dT). All possible variants of hydrogen bonding of base pairs of the same name were studied for each polymer. The maps of backbone chain geometrical existence were constructed. Conformational and helical parameters corresponding to local minima of conformational energy of “parallel” DNA helices, calculated at atom-atom approximation, were determined. The dependence of conformational energy on the base pair and on the hydrogen bond type was analysed. Two major conformational advantageous for “parallel” DNA's do not depend much on the hydrogen-bonded base pair type were indicated. One of them coincided with the conformational region typical for “antiparallel” DNA in particular for the B-form DNA Conformational energy of “parallel” DNA depends on the base pair type and for the most part is similar to the conformational energy of “antiparallel” B-DNA.     

Metal cation controls phosphate release in the myosin ATPase

Myosin is an enzyme that utilizes ATP to produce a conformational change generating a force. The kinetics of the myosin reverse recovery stroke depends on the metal cation complexed with ATP. The reverse recovery stroke is slow for MgATP and fast for MnATP. The metal ion coordinates the γ phosphate of ATP in the myosin active site. It is accepted that the reverse recovery stroke is correlated with the phosphate release; therefore, magnesium “holds” phosphate tighter than manganese. Magnesium and manganese are similar ions in terms of their chemical properties and the shell complexation; hence, we propose to use these ions to study the mechanism of the phosphate release. Analysis of octahedral complexes of magnesium and manganese show that the partial charge of magnesium is higher than that of manganese and the slightly larger size of manganese ion makes its ionic potential smaller. We hypothesize that electrostatics play a role in keeping and releasing the abstracted γ phosphate in the active site, and the stronger electric charge of magnesium ion holds γ phosphate tighter. We used stable myosin–nucleotide analog complex and Raman spectroscopy to examine the effect of the metal cation on the relative position of γ phosphate analog in the active site. We found that in the manganese complex, the γ phosphate analog is 0.01 nm further away from ADP than in the magnesium complex. We conclude that the ionic potential of the metal cation plays a role in the retention of the abstracted phosphate.     

The role of metal ions in the mechanism of action of hydrolytic metalloenzymes: Carbonic anhydrase

Metalloenzymes are among the most efficient enzymes. One of the mechanisms available to hydrolytic metalloenzymes consists of using the metal ion, which is embedded in the protein, as a carrier for hydroxide ions in neutral solution. Models for this mechanism are surveyed and analyzed from the point of view of the “charge effect”. The active center of carbonic anhydrase is compared to several of these models, and the similarities are pointed out. It is concluded that the “carrier for hydroxide ions” mechanism is the most plausible one for carbonic anhydrase. It is proposed that the metal ion also plays a role in the regeneration of the active center of the enzyme, i.e. the ionization of the metal-bound water molecule, after each turnover. Some general implications for the mechanism of action of other hydrolytic metalloenzymes are considered.     

On the link between conformational changes,ligand binding and heat capacity

BackgroundConformational changes coupled to ligand binding constitute the structural and energetics basis underlying cooperativity, allostery and, in general, protein regulation. These conformational rearrangements are associated with heat capacity changes. ITC is a unique technique for studying binding interactions because of the simultaneous determination of the binding affinity and enthalpy, and for providing the best estimates of binding heat capacity changes.Scope of reviewStill controversial issues in ligand binding are the discrimination between the “conformational selection model” and the “induced fit model”, and whether or not conformational changes lead to temperature dependent apparent binding heat capacities. The assessment of conformational changes associated with ligand binding by ITC is discussed. In addition, the “conformational selection” and “induced fit” models are reconciled, and discussed within the context of intrinsically (partially) unstructured proteins.Major conclusionsConformational equilibrium is a major contribution to binding heat capacity changes. A simple model may explain both conformational selection and induced fit scenarios. A temperature-independent binding heat capacity does not necessarily indicate absence of conformational changes upon ligand binding. ITC provides information on the energetics of conformational changes associated with ligand binding (and other possible additional coupled equilibria).General significancePreferential ligand binding to certain protein states leads to an equilibrium shift that is reflected in the coupling between ligand binding and additional equilibria. This represents the structural/energetic basis of the widespread dependence of ligand binding parameters on temperature, as well as pH, ionic strength and the concentration of other chemical species. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.