Changes In Ionic Conditions Research Articles

Staphylococcus aureus 30S Ribosomal Subunit Purification and Its Biochemical and Cryo-EM Analysis.

The ribosome is a complex cellular machinery whose solved structure allowed for an incredible leap in structural biology research. Different ions bind to the ribosome, stabilizing inter-subunit interfaces and structurally linking rRNAs, proteins, and ligands. Besides cations such as K + and Mg 2+ , polyamines are known to stabilize the folding of RNA and overall structure. The bacterial ribosome is composed of a small (30S) subunit containing the decoding center and a large (50S) subunit devoted to peptide bond formation. We have previously shown that the small ribosomal subunit of Staphylococcus aureus is sensitive to changes in ionic conditions and polyamines concentration. In particular, its decoding center, where mRNA codons and tRNA anticodons interact, is prone to structural deformations in the absence of spermidine. Here, we report a detailed protocol for the purification of the intact and functional 30S, achieved through specific ionic conditions and the addition of spermidine. Using this protocol, we obtained the cryo-electron microscopy (cryo-EM) structure of the 30S-mRNA complex from S. aureus at 3.6 Å resolution. The 30S-mRNA complex formation was verified by a toeprinting assay. In this article, we also include a description of toeprinting and cryo-EM protocols. The described protocols can be further used to study the process of translation regulation. Graphical abstract.

Charge regulation mechanism in end-tethered weak polyampholytes.

Weak polyampholytes, containing oppositely charged dissociable groups, are expected to be responsive to changes in ionic conditions. Here, we determine structural and thermodynamic properties, including the charged groups' degrees of dissociation, of end-tethered weak polyampholyte layers as a function of salt concentration, pH, and the solvent quality. For diblock weak polyampholytes grafted by their acidic blocks, we find that the acidic monomers increase their charge while the basic monomers decrease their charge with decreasing salt concentration for pH values less than the pKa value of both monomers and vice versa when the pH > pKa. This complex charge regulation occurs because the electrostatic attraction between oppositely charged blocks is stronger than the repulsion between monomers with the same charge in both good and poor solvents when the screening by salt ions is weak. This is evidenced by the retraction of the top block into the bottom layer. In the case of poor solvent conditions to the basic block (the top block), we find lateral segregation of basic monomers into micelles, forming a two-dimensional hexagonal pattern on the surface at intermediate and high pH values for monovalent salt concentrations from 0.01 to 0.1 M. When the solvent is poor to both blocks, we find lateral segregation of the grafted acidic block into lamellae with longitudinal undulations of low and high acidic monomer density. By exploiting weak block polyampholytes, our work expands the parameter space for creating responsive surfaces stable over a wide range of pH and salt concentration.

Free energy landscape of salt-actuated reconfigurable DNA nanodevices.

Achieving rapid, noninvasive actuation of DNA structures is critical to expanding the functionality of DNA nanotechnology. A promising actuation approach involves introducing multiple, short pairs of single-stranded DNA overhangs to components of the structure and triggering hybridization or dissociation of the overhangs via changes in solution ionic conditions to drive structural transitions. Here, we reveal the underlying basis of this new approach by computing via molecular simulations the free energy landscape of DNA origami hinges actuated between open and closed states. Our results reveal how the overhangs collectively introduce a sharp free-energy minimum at the closed state and a broad energy barrier between open and closed states and how changes in ionic conditions modulate these features of the landscape to drive actuation towards the open or closed state. We demonstrate the critical role played by hinge confinement in stabilizing the hybridized state of the overhangs and magnifying the energy barrier to dissociation. By analyzing how the distribution of overhangs and their length and sequence modulate the energy landscape, we obtain design rules for tuning the actuation behavior. The molecular insights obtained here should be applicable to a broad range of systems involving DNA hybridization within confined systems.

Molecular Mechanism of Interfacial Adsorption of Disordered Cytoplasmic Tail of Immune Receptors to Membrane

Important immune responses are linked to Src-mediated phosphorylation of cytoplasmic tyrosine-based motifs (ITAMs). However, the mechanism of how receptor ligation translates into ITAM phosphorylation remains elusive. Here, we use molecular dynamic simulations to explore the potential regulatory involvement of lipid membranes and their influence on the structure and behavior of the cytoplasmic portion of the CD3ɛ chain of the T-cell receptor. It has been hypothesized that the accessibility of ITAM motifs, such as those in the CD3ɛ cytoplasmic tail, can be blocked by ionic interactions between positively charged amino acids in receptor tails and negatively charged lipid head groups. This interesting hypothesis represents a previously unrecognized mechanism for control of receptor activation. Our simulations support the notion that the net charge of the lipids present in the membrane can affect peptide-membrane interactions. Results are consistent with experimental findings that show increase interfacial absorption in negatively charged lipid bilayer. Our simulations revealed the conformational variability of the disordered tail, which led to an additional focus on quantifying the interaction by free energy calculations, combined with long time-scale simulations using coarse-grained (CG) approaches. These studies will be extended to address how changes in ionic conditions can modulate phosphorylation of ITAM motifs and lead to regulation of activation of the TCR and other ITAM-bearing immunoreceptors.

A chamber for the perfusion of in vitro tissue with multiple solutions

There are currently no practical systems that allow extended regions (>5 mm(2)) of a tissue slice in vitro to be exposed, in isolation, to changes in ionic conditions or to pharmacological manipulation. Previous work has only achieved this at the expense of access to the tissue for recording electrodes. Here, we present a chamber that allows a tissue slice to be maintained in multiple solutions, at physiological temperatures, and preserves the ability to record from the slice. We demonstrate the effectiveness of the tissue bath with respect to minimizing the mixing of the solutions, maintaining the viability of the tissue, and preserving the ability to record from the slice simultaneously.

Assembly mechanism of recombinant spider silk proteins.

Spider silk threads are formed by the irreversible aggregation of silk proteins in a spinning duct with dimensions of only a few micrometers. Here, we present a microfluidic device in which engineered and recombinantly produced spider dragline silk proteins eADF3 (engineered Araneus diadematus fibroin) and eADF4 are assembled into fibers. Our approach allows the direct observation and identification of the essential parameters of dragline silk assembly. Changes in ionic conditions and pH result in aggregation of the two proteins. Assembly of eADF3 fibers was induced only in the presence of an elongational flow component. Strikingly, eADF4 formed fibers only in combination with eADF3. On the basis of these results, we propose a model for dragline silk aggregation and early steps of fiber assembly in the microscopic regime.

The internal equilibrium of the hairpin ribozyme: temperature, ion and pH effects

The hairpin ribozyme reversibly cleaves phosphodiesters of RNA substrates to generate products with 5′ hydroxyl and 2′,3′-cyclic phosphate termini. We previously found that the rate constant for ligation is tenfold faster than the rate constant for cleavage under standard conditions. The hammerhead ribozyme catalyzes the same reactions but is reported to favor cleavage relative to ligation by more than 100-fold under the same conditions. To explore the basis for this difference, we examined the influence of temperature, ions and pH on the hairpin ribozyme internal equilibrium. Under the same conditions, the loss of entropy associated with ligation is less for the hairpin than for the hammerhead ribozyme, consistent with the notion that a more rigid hairpin structure undergoes a smaller decrease in dynamics upon ligation than the more flexible hammerhead structure. Increased salt and reduced temperature shift the equilibrium toward ligation while pH has little effect, suggesting that conditions that stabilize RNA structure tend to promote ligation. The hairpin ribozyme appears to take up at least one tri- or divalent cation or two monovalent cations upon ligation. The efficiency with which different cations promote ligation depends strongly on valence and, less strongly, on ionic radius or electronegativity. This pattern of cation selectivity suggests that cations promote ligation through delocalized electrostatic shielding, perhaps interacting with a region of especially high charge density in the ligated ribozyme. Changes in ionic conditions produce large but compensating changes in enthalpy and entropy for cleavage and ligation. Thus, in addition to any increase in ribozyme dynamics associated with cleavage, re-organization of associated cations contributes significantly to hairpin ribozyme thermodynamics.

Macroscopic Na+ currents in the "Nonconducting" Shaker potassium channel mutant W434F.

C-type inactivation in Shaker potassium channels inhibits K+ permeation. The associated structural changes appear to involve the outer region of the pore. Recently, we have shown that C-type inactivation involves a change in the selectivity of the Shaker channel, such that C-type inactivated channels show maintained voltage-sensitive activation and deactivation of Na+ and Li+ currents in K+-free solutions, although they show no measurable ionic currents in physiological solutions. In addition, it appears that the effective block of ion conduction produced by the mutation W434F in the pore region may be associated with permanent C-type inactivation of W434F channels. These conclusions predict that permanently C-type inactivated W434F channels would also show Na+ and Li+ currents (in K+-free solutions) with kinetics similar to those seen in C-type-inactivated Shaker channels. This paper confirms that prediction and demonstrates that activation and deactivation parameters for this mutant can be obtained from macroscopic ionic current measurements. We also show that the prolonged Na+ tail currents typical of C-type inactivated channels involve an equivalent prolongation of the return of gating charge, thus demonstrating that the kinetics of gating charge return in W434F channels can be markedly altered by changes in ionic conditions.

Electrical impedance studies of nectarines during coolstorage and fruit ripening

The electrical impedance of ripe and unripe nectarine fruit was measured at harvest and after 3 and 8 weeks storage at 0°C, using frequencies of alternating current between 50 Hz and 0.1 MHz. The complex impedance was separated into its resistive and reactive components for both the electrodes and the fruit flesh. Electrode impedance was higher in stainless steel than in silver electrodes, and stainless steel electrodes were influenced by changes in ionic conditions. The resistance of whole fruit was lower than that of excised blocks of tissue from the same fruit. Changes in tissue resistance were greatest at low frequencies (50–100 Hz), with resistance increasing between 0 and 3 weeks storage and decreasing between 3 and 8 weeks storage. Changes in electrical resistance of nectarines are discussed, with the assumption that low frequency current travels via an extracellular pathway, and high frequency current travels via an intracellular pathway.

Colloid osmotic pressure of steer crystallins: Implications for the origin of the refractive index gradient and transparency of the lens

The osmotic behavior of soluble cortical and nuclear steer lens crystallins was characterized by secondary osmometry for several ionic strength and pH conditions. Osmotic pressure versus protein concentration relationships were measured for pressures up to 1.15 x 10(6) dyn cm-2. At low concentrations (< 0.2 g ml-1), the osmotic pressure increased linearly with pressure, whereas for concentrations above 0.2 g ml-1, the pressure rose more sharply, giving progressively larger changes in osmotic pressure with increasing crystallin concentration. At a given ionic strength and applied osmotic pressure, the nuclear proteins attained a higher protein concentration than did the cortical proteins. For example, at the highest osmotic pressure of 1.15 x 10(6) dyn cm-2 at pH 7.6 and 0.1 M ionic strength, the observed protein concentrations were 0.43 g ml-1 for the cortical proteins and 0.52 g ml-1 for the nuclear proteins. For both cortical and nuclear steer crystallins, the pressure rose more steeply with concentration than do pressures for calf crystallins described in the literature. The impact of these developmental differences in osmotic pressure on lens transparency is discussed. Both the nuclear and cortical crystallins exhibited ionic strength-dependent shifts in their pressure-concentration behavior. At 0.02 M ionic strength, higher pressures were observed, whereas at 0.4 M ionic strength lower pressures were observed for a given protein concentration. The crystallins were also found to equilibrate to different protein concentrations at a constant osmotic pressure and 0.1 M ionic strength over a pH range of 4-9, with a maximum concentration around pH 5 for the cortical crystallins and pH 6 for the nuclear crystallins. Thus, the adult bovine cortical and nuclear soluble lens extracts are different in their osmotic properties, reflecting underlying differences in protein composition. The results of the ionic strength and pH experiments suggest that hard-sphere, electrostatic, and Donnan forces contribute to the total colloid osmotic pressure of the lens crystallins. However, near physiologic pH and ionic strength the charges of the proteins are screened to the extent that the colloid osmotic pressure exhibits only minor changes for large changes in ionic conditions. The differences in the osmotic behavior of the cortical and nuclear proteins are consistent with a model where regional variations in the colloid osmotic properties of the proteins across the lens help support the radial refractive index gradient that is present in vertebrate lenses. The importance of a radial concentration gradient of metabolites is also discussed.

Studies on the stability of the higher-order structure of rat liver chromatin containing high-mobility-group proteins.

The stability of the higher-order structure of chromatin containing high-mobility-group (HMG) proteins has been studied in rat liver nuclei by mild micrococcal nuclease digestion at low temperature and fractionation by sucrose gradient centrifugation. Nuclei preparation and digestion, chromatin solubilization and analysis have been carried out in two ionic conditions, 140 mM and 40 mM monovalent cation concentration, avoiding drastic changes in ionic conditions and temperature during preparation and analysis. During the time course of digestion at 140 mM ionic strength a material stable at 80 S appears, whose DNA is cleaved at values around 12 nucleosomes. The distribution of HMG proteins in different chromatin fractions was analyzed by immunodot using antibodies elicited against proteins HMG-1, HMG-2, and HMG-14 and 17. It appears that these proteins have a distribution distinctly different from the bulk of chromatin. They are never found in the chromatin fragments that keep their internucleosomal interactions, indicating that these proteins tend to accumulate in points where the chromatin has a less stable structure.

Ionic changes in the mitotic apparatus at the metaphase/anaphase transition.

We have employed a series of permeant, nontoxic, fluorescent probes to detect changes in ionic conditions within the mitotic apparatus of living endosperm cells of Haemanthus during the transition from metaphase to anaphase. Fluorescence emission intensity measurements from the spindle for chlorotetracycline (CTC) decline before the onset of anaphase, indicating a reduction in the amount of membrane-associated Ca2+ and suggesting an efflux of Ca2+ from membrane compartments into the spindle. Subsequent to the onset of anaphase, we observe increases in fluorescence with both 8-anilino-1-naphthalene sulfonate (ANS) and 3,3'-dipentyl 2,2'-dioxacarbocyanine (diO-C5(3)), sensitive to cationic and anionic charges at membrane surfaces, respectively. The increases with ANS and diO-C5(3) suggest that redistributions of ions within the spindle accompany anaphase motion. During the metaphase/anaphase transition, spindle membrane content remains constant, as evidenced by unchanging fluorescence with the hydrophobic probe, N-phenyl-1-naphthylamine (NPN). Shifts in emission intensity from the nonspindle cytoplasm or from the spindle poles do not accompany the changes in fluorescence we observe in the spindle, suggesting that any ionic fluxes responsible for the changes in fluorescence are restricted to the spindle domain.

Binding of (acetyl-14C)trichodermin to the peptidyl transferase centre of eukaryotic ribosomes.

The antibiotic trichodermin, an inhibitor of the peptidyl transferase centre of eukaryotic ribosomes, was synthesized radioactively labeled. [acetyl-14C]Trichodermin (15.4 mCi/mmol) was obtained. [acetyl-14C]Trichodermin binds to ribosomes from yeast with a dissociation constant of 1.8 μM and to ribosomes of human tonsils with a dissociation constant of 0.67 μM but it does not bind to ribosomes of Escherichia coli. Similarly, the antibiotic binds to 60-S subunits derived from yeast ribosomes with a dissociation constant of 1.4 μM. A single-site interaction was observed in all cases. Trichodermin and anisomycin compete for binding to the peptidyl transferase centre. Although the activity of this centre is much affected by ionic conditions, the specific site of trichodermin interaction appears to be less sensitive since changes in ionic conditions, pH and ethanol concentration hardly modify the extent of antibiotic binding. Trichodermin binding to eukaryotic ribosomes is inhibited not only by anisomycin, but also by tenuazonic acid and a number of sesquiterpene antibiotics of the trichodermin group (tricho-dermol, trichothecin, fusarenon X and verrucarin A).