Combined effects of osmotic and hydrostatic pressure on multilamellar lipid membranes in the presence of PEG and trehalose.

We studied the interaction of lipid membranes with the disaccharide trehalose (TRH), which is known to stabilize biomembranes against various environmental stress factors. Generally, stress factors include low/high temperature, shear, osmotic and hydrostatic pressure. Small-angle X-ray-scattering was applied in combination with fluorescence spectroscopy and calorimetric measurements to get insights into the influence of trehalose on the supramolecular structure, hydration level, and elastic and thermodynamic properties as well as phase behavior of the model biomembrane DMPC, covering a large region of the temperature, osmotic and hydrostatic pressure phase space. We observed distinct effects of trehalose on the topology of the lipid's supramolecular structure. Trehalose, unlike osmotic pressure induced by polyethylene glycol, leads to a decrease of lamellar order and a swelling of multilamellar vesicles, which is attributable to direct interactions between the membrane and trehalose. Our results revealed a distinct biphasic concentration dependence of the observed effects of trehalose. While trehalose intercalates between the polar head groups at low concentrations, the effects after saturation are dominated by the exclusion of trehalose from the membrane surface.

Water-Mediated Protein-Protein Interactions at High Pressures are Controlled by a Deep-Sea Osmolyte.

The influence of natural cosolvent mixtures on the pressure-dependent structure and protein-protein interaction potential of dense protein solutions is studied and analyzed using small-angle X-ray scattering in combination with a liquid-state theoretical approach. The deep-sea osmolyte trimethylamine-N-oxide is shown to play a crucial and singular role in its ability to not only guarantee sustainability of the native protein's folded state under harsh environmental conditions, but it also controls water-mediated intermolecular interactions at high pressure, thereby preventing contact formation and hence aggregation of proteins.

On the Origin of Microtubules' High-Pressure Sensitivity.

For over 50 years, it has been known that the mitosis of eukaryotic cells is inhibited already at high hydrostatic pressure conditions of 30 MPa. This effect has been attributed to the disorganization of microtubules, the main component of the spindle apparatus. However, the structural details of the depolymerization and the origin of the pressure sensitivity have remained elusive. It has also been a puzzle how complex organisms could still successfully inhabit extreme high-pressure environments such as those encountered in the depth of oceans. We studied the pressure stability of microtubules at different structural levels and for distinct dynamic states using high-pressure Fourier-transform infrared spectroscopy and Synchrotron small-angle x-ray scattering. We show that microtubules are hardly stable under abyssal conditions, where pressures up to 100 MPa are reached. This high-pressure sensitivity can be mainly attributed to the internal voids and packing defects in the microtubules. In particular, we show that lateral and longitudinal contacts feature different pressure stabilities, and they define also the pressure stability of tubulin bundles. The intactness of both contact types is necessary for the functionality of microtubules in vivo. Despite being known to dynamically stabilize microtubules and prevent their depolymerization, we found that the anti-cancer drug taxol and the accessory protein MAP2c decrease the pressure stability of microtubule protofilaments. Moreover, we demonstrate that the cellular environment itself is a crowded place and accessory proteins can increase the pressure stability of microtubules and accelerate their otherwise highly pressure-sensitive de novo formation.

High-Pressure NMR and SAXS Reveals How Capping Modulates Folding Cooperativity of the pp32 Leucine-rich Repeat Protein.

Many repeat proteins contain capping motifs, which serve to shield the hydrophobic core from solvent and maintain structural integrity. While the role of capping motifs in enhancing the stability and structural integrity of repeat proteins is well documented, their contribution to folding cooperativity is not. Here we examined the role of capping motifs in defining the folding cooperativity of the leucine-rich repeat protein, pp32, by monitoring the pressure- and urea-induced unfolding of an N-terminal capping motif (N-cap) deletion mutant, pp32-∆N-cap, and a C-terminal capping motif destabilization mutant pp32-Y131F/D146L, using residue-specific NMR and small-angle X-ray scattering. Destabilization of the C-terminal capping motif resulted in higher cooperativity for the unfolding transition compared to wild-type pp32, as these mutations render the stability of the C-terminus similar to that of the rest of the protein. In contrast, deletion of the N-cap led to strong deviation from two-state unfolding. In both urea- and pressure-induced unfolding, residues in repeats 1-3 of pp32-ΔN-cap lost their native structure first, while the C-terminal half was more stable. The residue-specific free energy changes in all regions of pp32-ΔN-cap were larger in urea compared to high pressure, indicating a less cooperative destabilization by pressure. Moreover, in contrast to complete structural disruption of pp32-ΔN-cap at high urea concentration, its pressure unfolded state remained compact. The contrasting effects of the capping motifs on folding cooperativity arise from the differential local stabilities of pp32, whereas the contrasting effects of pressure and urea on the pp32-ΔN-cap variant arise from their distinct mechanisms of action.

Temperature and pressure limits of guanosine monophosphate self-assemblies.

Guanosine monophosphate, among the nucleotides, has the unique property to self-associate and form nanoscale cylinders consisting of hydrogen-bonded G-quartet disks, which are stacked on top of one another. Such self-assemblies describe not only the basic structural motif of G-quadruplexes formed by, e.g., telomeric DNA sequences, but are also interesting targets for supramolecular chemistry and nanotechnology. The G-quartet stacks serve as an excellent model to understand the fundamentals of their molecular self-association and to unveil their application spectrum. However, the thermodynamic stability of such self-assemblies over an extended temperature and pressure range is largely unexplored. Here, we report a combined FTIR and NMR study on the temperature and pressure stability of G-quartet stacks formed by disodium guanosine 5'-monophosphate (Na25'-GMP). We found that under abyssal conditions, where temperatures as low as 5 °C and pressures up to 1 kbar are reached, the self-association of Na25'-GMP is most favoured. Beyond those conditions, the G-quartet stacks dissociate laterally into monomer stacks without significantly changing the longitudinal dimension. Among the tested alkali cations, K+ is the most efficient one to elevate the temperature as well as the pressure limits of GMP self-assembly.

Exploring the effects of temperature and pressure on the structure and stability of a small RNA hairpin.

RNAs perform multiple vital roles within cells, including catalyzing biological reactions and expression of proteins. Small RNA hairpins (sRNAh) are the smallest functional entities of nucleic acids and are involved in various important biological functions such as ligand binding and tertiary folding initiation of proteins. We investigated the conformational and free energy landscape of the sRNAh gcUUCGgc over a wide range of temperatures and pressures using fluorescence resonance energy transfer, Fourier-transform infrared and UV/Vis spectroscopy as well as small-angle X-ray scattering on the unlabeled and/or fluorescently labeled sRNAh. The sRNAh shows a broad melting profile with continuous increase of unpaired conformations up to about 60°C. However, the sRNAh structure might not be fully unfolded at temperatures as high as 90°C and still comprise various partially unfolded compact conformations. Pressure up to 400MPa has a small effect on the base pairing and base stacking interactions of the sRNAh, indicating small conformational perturbations, only, which might originate from minor changes in packing and hydration of the RNA molecule upon compression. Pressurization at 70°C, i.e. at a temperature above the melting transition, has no significant effect on the conformational ensemble of the sRNAh, i.e., it does not promote formation of new native stem connections after thermal denaturation. Finally, we noticed that Cy3/Cy5 labeling of the sRNAh changes, probably via stacking interactions between the fluorescent dyes and the nucleotide rings, the stability of the sRNAh, thereby rendering FRET analysis of the conformational dynamics of such small RNA structure inappropriate.

Multiple Condensation Reactions Involving Pt(II) /Pd(II) -OH2 , Pt-NH3 , and Cytosine-NH2 Groups: New Twists in Cisplatin-Nucleobase Chemistry.

The coordination chemistry of the antitumor agent cisplatin and related complexes with DNA and its constituents, that is, the nucleobases, appears to be dominated by 1:1 and 1:2 adducts of the types cis-[Pta2 (nucleobase)X] and cis-[Pta2 (nucleobase)2 ] (a=NH3 or amine; a2 =diamine or diimine; X=Cl, OH or OH2 ). Here, we have studied the interactions of the putative 1:1 adducts cis-[Pta2 (1-MeC-N3)(OH2 )](2+) (with a=NH3 , a2 =2,2'-bpy (2,2'-bipyridine), 1-MeC=model nucleobase 1-methylcytosine) with additional cis-[Pt(NH3 )2 (OH2 )2 ](2+) or its kinetically superior analogues [Pd(en)(OH2 )2 ](2+) (en=ethylenediamine) and [Pd(2,2'-bpy)(OH2 )2 ](2+) . Depending upon the conditions applied different compounds of different nuclearity are formed. Without exception they represent condensation products of the components, containing µ-1-MeC-H , µ-OH(-) , as well as µ-NH2 (-) bridges. In the presence of Ag(+) ions, the isolated products in several cases display additionally Pt→Ag dative bonds. On the basis of the cytosine-containing structures established by X-ray crystallography, it is proposed that any of the feasible initial 1:1 nucleobase adducts of cisplatin could form dinuclear Pt complexes upon reaction with additional hydrolyzed cisplatin, thereby generating nucleobase adducts other than the presently established ones. Two findings appear to be of particular significance: First, hydrolyzed cisplatin can have a moderately accelerating effect on the formation of a secondary nucleobase product. Second, NH3 ligands of the cisplatin moiety can be converted into bridging amido ligands following condensation with the diaqua species of cisplatin.

Near-Surface and Bulk Behavior of Bicontinuous Microemulsions under High-Pressure Conditions.

The effect of hydrostatic pressure on the structure of a bicontinuous microemulsion in the presence of a solid interface has been studied by X-ray reflectometry and compared to the bulk behavior determined by small-angle X-ray scattering. Surface-induced lamellar ordering is observed close to the hydrophilic interface, which persists upon compression. The lamellar domains are compressed, but the correlation length of lamellar order does not change with pressure. SAXS measurements on the bulk microemulsion revealed an increased order upon pressurization. Although pressure can cause the formation of highly ordered lamellar phases from ordered bicontinuous cubic phases, such a scenario is not observed for the disordered analogue studied here. High pressure increases the stiffness of the interfacial surfactant layer, but this is not sufficient to overcome the loss in conformational entropy that would result from a transition to an ordered lamellar phase. Possible technological and biological implications of our results are briefly discussed.

Condensation agents determine the temperature-pressure stability of F-actin bundles.

Biological cells provide a large variety of rodlike filaments, including filamentous actin (F-actin), which can form meshworks and bundles. One key question remaining in the characterization of such network structures revolves around the temperature and pressure stabilities of these architectures as a way to understand why cells actively use proteins for forming them. The packing properties of F-actin in fascin- and Mg(2+) -induced bundles are compared, and significantly different pressure-temperature stabilities are observed because of marked differences in their nature of interaction, solvation, and packing efficiency. Moreover, differences are observed in their morphologies and disintegration scenarios. The pressure-induced dissociation of the actin bundles is reminiscent of a single unbinding transition as observed in other soft elastic manifolds.

Exploring the free energy and conformational landscape of tRNA at high temperature and pressure.

A combined temperature- and pressure-dependent study was employed to reveal the conformational and free-energy landscape of phenylalanine transfer RNA (tRNA(Phe) ), a known model for RNA function, to elucidate the features that are essential in determining its stability. These studies also help explore its structural properties under extreme environmental conditions, such as low/high temperatures and high pressures. To this end, fluorescence and FTIR spectroscopies, calorimetric and small-angle scattering measurements were carried out at different ion concentrations over a wide range of temperatures and pressures up to several hundred MPa. Compared with the pronounced temperature effect, the pressure-dependent structural changes of tRNA(Phe) are small. A maximum of only 15 % unpaired bases is observed upon pressurization up to 1 GPa. RNA unfolding differs not only from protein unfolding, but also from DNA melting. Its pressure stability seems to be similar to that of noncanonical DNA structures.

Productive encounter: molecularly imprinted nanoparticles prepared using magnetic templates.

Synthesis of core-shell nanoparticles by surface initiated reversible addition fragmentation chain transfer polymerization in presence of a chiral template conjugated to magnetic nanoparticles is reported. The approach leads to imprinted nanoparticles featuring enantioselectivity and enhanced affinity compared to nanoparticles prepared using free template.

Prebiotic cell membranes that survive extreme environmental pressure conditions.

Attractive candidates for compartmentalizing prebiotic cells are membranes comprised of single-chain fatty acids. It is generally believed that life may have originated in the depth of the protoocean, that is, under high hydrostatic pressure conditions, but the structure and physical-chemical properties of prebiotic membranes under such conditions have not yet been explored. We report the temperature- and pressure-dependent properties of membranes composed of prebiotically highly-plausible lipids and demonstrate that prebiotic membranes could not only withstand extreme temperatures, but also serve as robust models of protocells operating in extreme pressure environments. We show that pressure not only increases the stability of vesicular systems but also limits their flexibility and permeability to solutes, while still keeping the membrane in an overall fluid-like and thus functional state.