Structural Determinants of the Mechanical Stability of α-Catenin

α-Catenin plays a crucial role in cadherin-mediated adhesion by binding to β-catenin, F-actin, and vinculin, and its dysfunction is linked to a variety of cancers and developmental disorders. As a mechanotransducer in the cadherin complex at intercellular adhesions, mechanical and force-sensing properties of α-catenin are critical to its proper function. Biochemical data suggest that α-catenin adopts an autoinhibitory conformation, in the absence of junctional tension, and biophysical studies have shown that α-catenin is activated in a tension-dependent manner that in turn results in the recruitment of vinculin to strengthen the cadherin complex/F-actin linkage. However, the molecular switch mechanism from autoinhibited to the activated state remains unknown for α-catenin. Here, based on the results of an aggregate of 3 μs of molecular dynamics simulations, we have identified a dynamic salt-bridge network within the core M region of α-catenin that may be the structural determinant of the stability of the autoinhibitory conformation. According to our constant-force steered molecular dynamics simulations, the reorientation of the MII/MIII subdomains under force may constitute an initial step along the transition pathway. The simulations also suggest that the vinculin-binding domain (subdomain MI) is intrinsically much less stable than the other two subdomains in the M region (MII and MIII). Our findings reveal several key insights toward a complete understanding of the multistaged, force-induced conformational transition of α-catenin to the activated conformation.     

Stability of toxin gene proportion in red-pigmented populations of the cyanobacterium <Emphasis Type="Italic">Planktothrix</Emphasis> during 29 years of re-oligotrophication of Lake Zürich

Background

Harmful algal blooms deteriorate the services of aquatic ecosystems. They are often formed by cyanobacteria composed of genotypes able to produce a certain toxin, for example, the hepatotoxin microcystin (MC), but also of nontoxic genotypes that either carry mutations in the genes encoding toxin synthesis or that lost those genes during evolution. In general, cyanobacterial blooms are favored by eutrophication. Very little is known about the stability of the toxic/nontoxic genotype composition during trophic change.

Results

Archived samples of preserved phytoplankton on filters from aquatic ecosystems that underwent changes in the trophic state provide a so far unrealized possibility to analyze the response of toxic/nontoxic genotype composition to the environment. During a period of 29 years of re-oligotrophication of the deep, physically stratified Lake Zürich (1980 to 2008), the population of the stratifying cyanobacterium Planktothrix was at a minimum during the most eutrophic years (1980 to 1984), but increased and dominated the phytoplankton during the past two decades. Quantitative polymerase chain reaction revealed that during the whole observation period the proportion of the toxic genotype was strikingly stable, that is, close to 100%. Inactive MC genotypes carrying mutations within the MC synthesis genes never became abundant. Unexpectedly, a nontoxic genotype, which lost its MC genes during evolution, and which could be shown to be dominant under eutrophic conditions in shallow polymictic lakes, also co-occurred in Lake Zürich but was never abundant. As it is most likely that this nontoxic genotype contains relatively weak gas vesicles unable to withstand the high water pressure in deep lakes, it is concluded that regular deep mixing selectively reduced its abundance through the destruction of gas vesicles.

Conclusions

The stability in toxic genotype dominance gives evidence for the adaptation to deep mixing of a genotype that retained the MC gene cluster during evolution. Such a long-term dominance of a toxic genotype draws attention to the need to integrate phylogenetics into ecological research as well as ecosystem management.

     

Stability of fungal α-amylase in sodium dodecylsulfate

Unfolding of a fungal -amylase in aqueous sodium dodecylsulfate (SDS) solution was examined by SDS-polyacrylamide gel electrophoresis (PAGE). When the -amylase was incubated with 1% SDS at room temperature and subjected to SDS-PAGE, it showed a much higher mobility than expected from the molecular weight. Circular dichroic and gel filtration analyses indicated that the protein is apparently in the native conformation upon incubation with 1% SDS. When the protein was heated in the presence of 1% SDS at 90°C for 10 min, it had a lower mobility in SDS-PAGE and showed characteristics of an unfolded protein by circular dichroism and gel filtration. The melting temperatures of the protein were determined in the absence and presence of SDS by incubating it for 10 min at various temperatures. The melting temperatures were 70, 55, and 49°C in the presence of 0, 1, and 2% SDS, respectively. The observed small shift of the melting temperatures by SDS suggests that the destabilizing action of SDS on the -amylase is weak. However, the unfolding in SDS is not reversible process, since prolonged incubation of the protein with 1% SDS at 50°C gradually increased the amount of unfolded protein. This indicates that the SDS-induced unfolding of the -amylase is a slow process.     

Stability of symmetric idiotypic networks—a critique of Hoffmann's analysis

Hoffmann (1982) analysed a very simple model of suppressive idiotypic immune networks and showed that idiotypic interactions are stabilizing. He concluded that immune networks provide a counterexample to the general analysis of large dynamic systems (Gardner and Ashby, 1970; May, 1972). The latter is often verbalized as: an increase in size and/or connectivity decreases the system stability. We here analyse this apparent contradiction by extending the Hoffmann model (with a decay term), and comparing it to an ecological model that was used as a paradigm in the general analysis. Our analysis confirms that the neighbourhood stability of such idiotypic networks increases with connectivity and/or size. However, the contradiction is one of interpretation, and is not due to exceptional properties of immune networks. The contradiction is caused by the awkward normalization used in the general analysis.     

Thermal Stability Investigation and the Kinetic Study of Folnak<Superscript>®</Superscript> Degradation Process Under Nonisothermal Conditions

The nonisothermal degradation process of Folnak^® drug samples was investigated by simultaneous thermogravimetric and differential thermal analysis in the temperature range from an ambient one up to 810°C. It was established that the degradation proceeds through the five degradation stages (designated as I, II, III, IV, and V), which include: the dehydration (I), the melting process of excipients (II), as well as the decomposition of folic acid (III), corn starch (IV), and saccharose (V), respectively. It was established that the presented excipients show a different behavior from that of the pure materials. During degradation, all excipients increase their thermal stability, and some kind of solid–solid and/or solid–gas interaction occurs. The kinetic parameters and reaction mechanism for the folic acid decomposition were established using different calculation procedures. It was concluded that the folic acid decomposition mechanism cannot be explained by the simple reaction order (ROn) model (n?=?1) but with the complex reaction mechanism which includes the higher reaction orders (RO, n?>?1), with average value of <n?>?=?1.91. The isothermal predictions of the third (III) degradation stage of Folnak^® sample, at four different temperatures (T _iso?=?180°C, 200°C, 220°C, and 260°C), were established. It was concluded that the shapes of the isothermal conversion curves at lower temperatures (180–200°C) were similar, whereas became more complex with further temperature increase due to the pterin and p-amino benzoic acid decomposition behavior, which brings the additional complexity in the overall folic acid decomposition process.     

Effect of pH and Glucose on the Stability of α-lactalbumin

The objective of this study is to understand the influence of pH and effect of cosolvent (glucose) on the stabilization of bovine α-lactalbumin by using ultrasonic techniques. Values of density, ultrasonic velocity and viscosity were measured for bovine α-lactalbumin (5 mg/ml) dissolved in phosphate buffer (pH 2, 5, 7, 9 and 12) solutions mixed with and without the cosolvent at 30 °C. These measurements were used to calculate few thermo-acoustical parameters such as adiabatic compressibility, intermolecular free length, acoustic impedance, relaxation time, relative association constant, the partial apparent specific volume and the partial apparent specific adiabatic compressibility for the said systems. The obtained results revealed a strong comparison between the effects of acidic and alkaline pH values on protein denaturation, i.e., the acidic pH are instantaneous and are of less magnitude whereas alkaline pH are slower but sharper. Further the present study supports the fact that the presence of glucose stabilizes α-lactalbumin against denaturation due to pH variation, which may be due to the strengthening of non-covalent interactions and the steric exclusion effect.     

The α-Helix of Ribonuclease T1as an Independent Stability Unit: Direct Comparison of Peptide and Protein Stability

Measurements of the change in conformational stability, Δ(ΔG), upon mutation of two acidic residues at the C terminus of the helix of ribonuclease T₁have recently been reported. Here, we investigate peptides based on the sequence of the helix with the same mutations: Glu28 replaced with Gln, Asp29 replaced with Asn, and the double mutant. In addition, the mutant Lys25 to Gln was studied. Changes in helix content of the peptides with pH confirm the conclusion found in the intact protein, that the charged forms of the acidic residues destabilize the protein by destabilizing the helix. The pH-dependence of the change in confor mational free energy for the peptides and mutant proteins show fair correspondence for D29N and the double mutant. The mutants E28Q and K25Q, on the other hand, give striking agreement between the protein and peptide systems. This agreement suggests that the helix of ribonuclease T₁behaves as an independently stabilized structural unit of the intact protein and that stabilization of the helical form of the peptide is mirrored in the protein.     

Stability of RNA quadruplex in open reading frame determines proteolysis of human estrogen receptor α

mRNAs encodes not only information that determines amino acid sequences but also additional layers of information that regulate the translational processes. Notably, translational halt at specific position caused by rare codons or stable RNA structures is one of the potential factors regulating the protein expressions and structures. In this study, a quadruplex-forming potential (QFP) sequence derived from an open reading frame of human estrogen receptor α (hERα) mRNA was revealed to form parallel G-quadruplex and halt the translation elongation in vitro. Moreover, when the full-length hERα and variants containing synonymous mutations in the QFP sequence were expressed in cells, translation products cleaved at specific site were observed in quantities dependent on the thermodynamic stability of the G-quadruplexes. These results suggest that the G-quadruplex formation in the coding region of the hERα mRNA impacts folding and proteolysis of hERα protein by slowing down or temporarily stalling the translation elongation.     

Stability of immobilized α-chymotrypsin in supercritical carbon dioxide

Summary Inactivation of immobilized -chymotrypsin in supercritical carbon dioxide was with a first-order kinetic behaviour. The increase in either the pressure or the temperature of the fluid enhanced the inactivation process of the enzyme. The fluid density was shown as a key parameter on the enzyme stability, enhancing the half-life time proportionally to the physical phase of CO₂, as follows: liquid > supercritical > gas. However, the number of pressurization/depressurization cycles, and the water content of the derivative increased greatly the loss of activity.     

Solvation Energy and Thermal Stability of Hydrophilization-Modified α-Chymotrypsin

As reported in the literature [Mozhaev et al. (1988), Eur. J. Biochem. 173, 147–154], when a series of modifiers, especially the cyclic anhydrides of pyromellitic and mellitic acids, are introduced into each lysine located in the -chymotrypsin (CT) surface, a substantial hydrophilization of the enzyme surface can occur and remarkable stabilization effects of modified enzymes can be obtained. In this paper, four models are applied to calculate the solvation energy of native and the modified CT based on their tertiary structures, which can be built by the CVFF force field. Analyzing the relationship between the solvation energy and the thermal stability in detail, we find that the results of three solvation energy models (Ooi model, WE-1 model, and WE-2 model) can be used to illustrate the relative stability among these enzymes qualitatively. The present study should be of practical value as well as of some theoretical interest.     

Stability of Schiff bases of amino acids and pyridoxal-5′-phosphate

Summary Stability of Schiff bases from Pyridoxal-5-phosphate and- and non-amino acids and amines have been studied in a wide range of pH. Furthermore the transamination process for the PLP-serine Schiff base and the cyclization reaction of PLP-histidine Schiff base have also been studied.Results show that the-position on carboxyl group of amino acids plays an important role on the mechanism of hydrolysis of imine bond. Absence of ionic groups in the surroundings of that bond seems to be an important fact of stability.In the transamination reaction, the rate-determining step is the isomerization of the Schiff base to ketoimine, since the rate constants for disappearance of Schiff base coincide with the rate constants for PMP formation. This process is catalyzed by the OH^–/H₂O system and the monoprotonated amino acid.     

Effects of Turn Stability and Side-Chain Hydrophobicity on the Folding of β-Structures

Key elements of β-structure folding include hydrophobic core collapse, turn formation, and assembly of backbone hydrogen bonds. In the present folding simulations of several β-hairpins and β-sheets (peptide 1, protein G B1 domain peptide, TRPZIP2, TRPZIP4, 20mer, and 20mer^DP6D), the folding free-energy landscape as a function of several reaction coordinates corresponding to the three key elements indicates apparent dependence on turn stability and side-chain hydrophobicity, which demonstrates different folding mechanisms of similar β-structures of varied sequences. Turn stability is found to be the key factor in determining the formation order of the three structural elements in the folding of β-structures. Moreover, turn stability and side-chain hydrophobicity both affect the stability of backbone hydrogen bonds. The three-stranded β-sheets fold through a three-state transition in which the formation of one hairpin always takes precedence over the other. The different stabilities of two anti-parallel hairpins in each three-stranded β-sheet are shown to correlate well with the different levels of their hydrophobic interactions.     

Implication of C-Terminal Deletion on the Structure and Stability of Bovine β-casein

Bovine β-casein (β-CN) with its C-terminal truncated by chymosin digestion, β-CN-(f1-192), was examined and characterized using circular dichroism (CD) under various temperature conditions. CONTIN/LL analysis of the CD data revealed significant secondary structure disruption in β-CN-(f1-192) relative to its parent protein,β-CN, in the temperature range (5° to 70°C) studied. Near-UV CD spectra indicated significant temperature dependent structural changes. Analytical ultracentrifugation results showed significant reduction but not complete abolishment of self-association in β-CN-(f1-192) compared to whole β-casein at 2°–37°C. Furthermore, binding experiments with the common hydrophobic probe – 8-anilino-1- naphthalene sulfonic acid (ANS) illustrated that β-CN-(f1-192) is nearly incapable of binding to ANS relative to whole β-CN, suggesting a nearly complete open overall tertiary structure brought about by the C-terminal truncation. It has been demonstrated clearly that the tail peptide β-CN-(f193-209) is important in maintaining the hydrophobic core of β-CN but the residual association observed argues for a minor role for other sites as well.     

Stability and electronic properties of praseodymium-doped silicon clusters PrSin (<Emphasis Type="Italic">n</Emphasis> = 12–21)

The neutral PrSi _n (n = 12–21) species considering various spin configurations were systematically studied using PBE0 and B3LYP schemes in combination with relativistic small-core potentials (ECP28MWB) for Pr atoms and cc-pVTZ basis set for Si atoms. The total energy, growth-pattern, equilibrium geometry, relative stability, hardness, charge transfer, and magnetic moments are calculated and discussed. The results reveal that when n < 20, the ground-state structure of PrSi _n evaluated to be prolate clusters. Starting from n = 20, the ground-state structures of PrSi _n are evaluated to be endohedral cagelike clusters. Although the relative stabilities based on various binding energies and different functional is different from each other, the consensus is that the PrSi₁₃, PrSi₁₆, PrSi₁₈, and PrSi₂₀ are more stable than the others, especially the PrSi₂₀. Analyses of hardness show that introducing Pr into Si _n (n = 12–21) elevates the photochemical sensitivity, especially for PrSi₂₀. Calculated result of magnetic moment and charge transfer shows that the 4f electrons of Pr in the clusters are changed, especially in endohedral structures such as PrSi₂₀, in which one electron transfers from 4f to 5d orbital. That is, the 4f electron of Pr in the clusters participates in bonding. The way to participate in bonding is that a 4f electron transfers to 5d orbital. Although the 4f electron of Pr atom participates in bonding, the total magnetic moment of PrSi _n is equal to that of isolated Pr atom. The charge always transfers from Pr atom to Si _n cluster for the ground state structures of PrSi_n (n = 12–19), but charge transfer is reverse for n ≥ 20. The largest charge transfer for endohedral structure reveals that the bonding between Pr and Si _n is ionic in nature and very strong. The fullerenelike structure of PrSi₂₀ is the most stable among all of these clusters and can act as the building blocks for novel functional nanotubes.     

Analyzing Thermal Stability of Cell Membrane of Salmonella Using Time-Multiplexed Impedance Sensing

Heat treatment is one of the most widely used methods for inactivation of bacteria in food products. Heat-induced loss of bacterial viability has been variously attributed to protein denaturation, oxidative stress, or membrane leakage; indeed, it is likely to involve a combination of these processes. We examine the effect of mild heat stress (50–55°C for ≤12 min) on cell permeability by directly measuring the electrical conductance of samples of Salmonella enterica serovar Typhimurium to answer a fundamental biophysical question, namely, how bacteria die under mild heat stress. Our results show that when exposed to heat shock, the cell membrane is damaged and cells die mainly due to the leakage of small cytoplasmic species to the surrounding media without lysis (confirmed by fluorescent imaging). We measured the conductance change, ΔY, of wild-type versus genetically modified heat-resistant (HR) cells in response to pulse and ramp heating profiles with different thermal time constants. In addition, we developed a phenomenological model to correlate the membrane damage, cytoplasmic leakage, and cell viability. This model traces the differential viability and ΔY of wild-type and HR cells to the difference in the effective activation energies needed to permeabilize the cells, implying that HR cells are characterized by stronger lateral interactions between molecules, such as lipids, in their cell envelope.