Tetravalent Spermine Research Articles

The Effect of Multivalent Cations on Microtubule-Protein Tau Ordering

In previous studies, it was found that paclitaxel-stabilized microtubules (MTs), hollow protein nanotubes comprised of assembled αβ-tubulin heterodimers, spontaneously assemble into bundles above a critical concentration (∼1.5 mM Spermine) of tetravalent spermine (PNAS 2004, 101, 16099). Further, at concentrations of spermine several-fold higher (∼10 mM Spermine), paclitaxel-stabilized MT bundles (BMT) quickly become unstable and undergo a shape transformation to bundles of inverted tubulin tubules (BITT), the outside surface of which corresponds to the inner surface of the BMT tubules (Nature Materials 2014, 13, 195). Here we will report on our current study of protein Tau-microtubule ordering (in an active system) in the absence of paclitaxel and, in particular, the effect of biological cations on microtubule self-assembly using transmission electron microscopy (TEM) and synchrotron small-angle X-ray scattering (SAXS).

Condensation of Nucleic Acids by Multivalent Ions: Sequence Dependence and the Curious Case of RNA

Highly charged nucleic acid (NA) molecules repel each other in monovalent salt solutions, yet can be condensed by multivalent ions into structured aggregates. This phenomenon is of great significance to biology, biotechnology and medicine. Surprisingly, recent experiments (Physical Review Letters 2011, 106, 108101; Nucleic Acids Research 2014, 42, 10823) demonstrate that short double-stranded RNA helices resist condensation under conditions where short DNA duplexes condense readily. Existing theoretical models of the NA condensation can not satisfactorily explain this striking differences in RNA vs. DNA condensation behavior. We combine experiment, atomistic molecular dynamics simulations and theory to propose a mechanism that connects the observed variations in condensation of short NA duplexes by trivalent cobalt hexammine and tetravalent spermine cations with a spatial variation of bound counterion charge atthe NA duplex surface. Specifically, NA condensation propensity is determined by the fraction of neutralizing multivalent counterions charge localized at the external (outermost) surface of the double-helix. A simple model is developed that provides semi-quantitative agreement with experiment, including subtle sequence effects on condensation propensity. Latest developments and experimental verification of the model will also be discussed.

Transformation of taxol-stabilized microtubules into inverted tubulin tubules triggered by a tubulin conformation switch

Bundles of taxol-stabilized microtubules (MTs) – hollow tubules comprised of assembled αβ-tubulin heterodimers – spontaneously assemble above a critical concentration of tetravalent spermine and are stable over long times at room temperature. Here we report that at concentrations of spermine several-fold higher the MT bundles (BMT) quickly become unstable and undergo a shape transformation to bundles of inverted tubulin tubules (BITT), the outside surface of which corresponds to the inner surface of the BMT tubules. Using transmission electron microscopy and synchrotron small-angle x-ray scattering, we quantitatively determined both the nature of the BMT to BITT transformation pathway, which results from a spermine-triggered conformation switch from straight to curved in the constituent taxol-stabilized tubulin oligomers, and the structure of the BITT phase, which is formed of tubules of helical tubulin oligomers. Inverted tubulin tubules provide a platform for studies requiring exposure and availability of the inside, luminal surface of MTs to MT-targeted-drugs and MT-associated-proteins.

Physical Determinants of Strong Voltage Dependence of K+ Channel Block

Many pharmacological agents, as well as some endogenous biological molecules, act by blocking ion channels in a strongly voltage-dependent manner. In retrospect, the first and most dramatic example of such action is the “anomalous” voltage dependence of inwardly rectifying K+ (Kir) conductance discovered by Bernard Katz six decades ago. He observed that, contrary to the classic voltage-gated K+ conductance, the Kir conductance tends to zero with membrane depolarization but increases with hyperpolarization. In intact cells and within physiological voltage ranges, certain Kir channels are as steeply voltage dependent as Kv channels yet, unlike Kv channels, they have no inherent voltage sensors: the observed voltage sensitivity instead reflects voltage-dependent block of their ion pore by intracellular cations such as the polyamine spermine. Our group has proposed that the high valence associated with block of strong rectifiers primarily reflects the movement, not of tetravalent spermine itself, but of five K+ ions displaced by spermine across the steep electric field in the narrow K+ selectivity filter. We will present experimental evidence for key requirements of this blocker-K+ displacement model, and will discuss the essential features that render a pore-blocking process strongly voltage dependent.

Abrupt Transition from a Free, Repulsive to a Condensed, Attractive DNA Phase, Induced by Multivalent Polyamine Cations

We have investigated the energetics of DNA condensation by multivalent polyamine cations. Solution small angle x-ray scattering was used to monitor interactions between short 25 base pair dsDNA strands in the free supernatant DNA phase that coexists with the condensed DNA phase. Interestingly, when tetravalent spermine is used, significant inter-DNA repulsion is observed in the free phase, in contrast with the presumed inter-DNA attraction in the coexisting condensed phase. DNA condensation thus appears to be a discrete, first-order-like, transition from a repulsive gaseous to an attractive condensed solid phase, in accord with the reported all-or-none condensation of giant DNA. We further quantify the electrostatic repulsive potentials in the free DNA phase and estimate the number of additional spermine cations that bind to DNA upon condensation.

Self-Assembly of Lyotropic Chromonic Liquid Crystal Sunset Yellow and Effects of Ionic Additives

Lyotropic chromonic liquid crystals (LCLCs) are formed by molecules with ionic groups at the periphery that associate into stacks through noncovalent self-assembly while in water. The very existence of the nematic (N) phase in the typical LCLC, the dye Sunset Yellow (SSY) is a puzzle, as the correlation length associated with the stacking, as measured in the X-ray experiments, is too short to explain the orientational order by the Onsager model. We propose that the aggregates can be more complex than simple rods and contain "stacking faults" such as junctions with a shift of neighboring molecules, 3-fold junctions, etc. We study how ionic additives, such as salts of different valency and pH-altering agents, alter the N phase of SSY purified by recrystallization. The additives induce two general trends: (a) stabilization of the N phase, caused by the mono and divalent salts (such as NaCl), and evidenced by the increase of the N-to-I transition temperature and the correlation length; (b) suppression of the N phase manifested in the decrease of the N-to-I transition temperature and in separation of the N phase into a more densely packed N phase or the columnar (C) phase, coexisting with a less condensed I phase. The scenario (b) can be triggered by simply increasing pH (adding NaOH). The effects produced by tetravalent spermine fall mostly into the category (b), but the detail depends on whether this additive is in its salt form or a free base form. The base form causes changes through changes in pH and possible excluded volume effects whereas the salt form might disrupt the structure of SSY aggregates.

Reduction of DNA contamination in RNA samples for reverse transcription-polymerase chain reaction using selective precipitation by compaction agents

An important problem in measurement of messenger RNA (mRNA) levels by reverse transcription-polymerase chain reaction (RT-PCR) is DNA contamination, which can produce artifactually increased mRNA concentration. Current methods to eliminate contaminating DNA can compromise the integrity of the RNA, are time-consuming, and/or are hazardous. We present a rapid, nuclease-free, and cost-effective method of eliminating contaminating DNA in RNA samples using selective precipitation by compaction agents. Compaction agents are cationic molecules that bind to double-stranded nucleic acids, driven by electrostatic interactions and steric complementarity. The effectiveness and DNA selectivity of six compaction agents were investigated: trivalent spermidine, Triquat A, and Triquat 7; tetravalent spermine and Quatro-quat; and hexavalent Quatro-diquat. Effectiveness was measured initially by supernatant UV absorbance after precipitation of salmon sperm DNA. Effectiveness and selectivity were then investigated using differences in RT-PCR C t values with synthetic mixtures of human genomic DNA and total RNA and with total RNA isolated from cells. With 500 μM spermidine or Triquat A, the supernatant DNA could not be detected up to 40 cycles of PCR ( C t ⩾ 12.6), whereas the C t for the mRNA was increased by only five cycles. Therefore, spermidine and Triquat A each show strong DNA selectivity and could be used to eliminate contaminating DNA in measurements of mRNA.

Phospholipid-Based Artificial Viruses Assembled by Multivalent Cations

Self-assembled DNA delivery systems based on cationic lipids are simple to produce and weakly hazardous in comparison with viral vectors, but possess a significant toxicity at high doses. Phospholipids are in contrast intrinsically safe; yet their association with DNA is problematic because of unfavorable electrostatic interactions. We achieve the phospholipid-DNA complexation through the like-charge attraction induced by cations. Monovalent cations are inappropriate due to their poor binding affinity with lipids as inferred from electrophoretic mobility, whereas x-ray diffractions reveal that with multivalent cations, DNA is complexed within an inverted hexagonal liquid-crystalline phase. Coarse-grained Monte Carlo simulations confirm the self-assembly of a DNA rod wrapped into a lipid layer with cations in between acting as molecular glue. Transfection experiments performed with Ca2+ and La3+ demonstrate efficiencies surpassing those obtained with optimized cationic DOTAP-based systems, while preserving the viability of cells. Inspired by bacteriophages that resort to polycations to compact their genetic materials, complexes assembled with tetravalent spermine achieve unprecedented transfection efficiencies for phospholipids. Influence of complex growth time, lipid/DNA mass ratio, and ion concentration are examined. These complexes may initiate new developments for nontoxic gene delivery and fundamental studies of biological self-assembly.

Dielectric Control of Counterion-Induced Single-Chain Folding Transition of DNA

In the presence of condensing agents, single chains of giant double-stranded DNA undergo a first-order phase transition between an elongated coil state and a folded compact state. To connect this like-charged attraction phenomenon to counterion condensation, we performed a series of single-chain experiments on aqueous solutions of DNA, where we varied the extent of counterion condensation by varying the relative dielectric constant epsilon(r) from 80 to 170. Single-chain observations of changes in the conformation of giant DNA were performed by transmission electron microscopy and fluorescence microscopy, with tetravalent spermine (SPM(4+)) as a condensing agent. At a fixed dielectric constant, single DNA chains fold into a compact state upon the addition of spermine, whereas at a constant spermine concentration single DNA chains unfold with an increase in epsilon(r). In both cases, the transition is largely discrete at the level of single chains. We found that the critical concentration of spermine necessary to induce the single-chain folding transition increases exponentially as the dielectric constant increases, corresponding to 87-88% of the DNA charge neutralized at the onset of the transition. We also observed that the toroidal morphology of compact DNA partially unfolds when epsilon(r) is increased.

Myristoylated Alanine-rich C Kinase Substrate (MARCKS) Produces Reversible Inhibition of Phospholipase C by Sequestering Phosphatidylinositol 4,5-Bisphosphate in Lateral Domains

The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a major protein kinase C (PKC) substrate in many different cell types. MARCKS is bound to the plasma membrane, and several recent studies suggest that this binding requires both hydrophobic insertion of its myristate chain into the bilayer and electrostatic interaction of its cluster of basic residues with acidic lipids. Phosphorylation of MARCKS by PKC introduces negative charges into the basic cluster, reducing its electrostatic interaction with acidic lipids and producing translocation of MARCKS from membrane to cytoplasm. The present study shows that physiological concentrations of MARCKS (<10 microM) inhibit phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in phospholipid vesicles. A peptide corresponding to the basic cluster, MARCKS(151-175), produces a similar inhibition, which was observed with both PLC-delta1 and -beta1. Direct fluorescence microscopy observations demonstrate that the MARCKS peptide forms lateral domains enriched in the acidic lipids phosphatidylserine and PIP2 but not PLC, which accounts for the observed inhibition of PIP2 hydrolysis. Phosphorylation of MARCKS(151-175) by PKC releases the inhibition and allows PLC to produce a burst of inositol 1,4, 5-trisphosphate and diacylglycerol.

Multiple effects of ions on ATP: l-arginine and ATP: Creatine phosphotransferases

1. 1. Cations inactivate Sipunculus arginine kinase (mol. wt 84 000) (ATP: l-arginine phosphotransferase, EC 2.7.3.3). The order of increasing effectiveness is K +, NH 4 +, Li +, guanidinium cation, tetravalent spermine action. The action of lithium acetate and guanidine · HCl is expressed by a two-step inhibition: an initial immediate inhibition which is of the non-competitive type with respect to l-arginine and Mg 2+- ATP, and a slower inactivation. While the first stage can be observed at 10°C, the rate of the secondary one increases with temperature. This inhibition is similar to that observed with anions. The modification of the protein absorption spectrum which characterizes the action of anions is displayed with guanidine · HCl and spermine · 4HCl but not with K +, NH 4 +, Li +. 2. 2. The effects of KCl on Sipunculus arginine kinase in H 2O and in 2H 2O are qualitatively similar but quantitatively different. In 2H 2O the decrease of V by KCl is greater than in H 2O. However, the modification of the protein absorption spectrum is less sharp in 2H 2O than in H 2O. 3. 3. Rabbit muscle creatine kinase (ATP:creatine phosphotransferase, EC 2.7.3.2) shows a susceptibility towards anions and cations comparable to that of Sipunculus arginine kinase. The order of effectiveness of anions and cations on both enzymes is the same. The kinetic behaviour of the action of Cl − is identical for both enzymes. Nevertheless no modification of the protein absorbance spectrum is observed by the interaction of Cl −.