Hydrophobic core variant ubiquitin forms a molten globule conformation at acidic pH

The conformational properties of hydrophobic core variant ubiquitin (Val26 to Ala mutation) in an acidic solution were studied. The intrinsic tryptophan fluorescence emission spectrum, far-UV and near-UV circular dichroic spectra, the fluorescence emission spectrum of 8-anilinonaphthalene-1-sulfonic acid in the presence of V26A ubiquitin, and urea-induced unfolding measurements indicate this variant ubiquitin to be in the partially folded molten globule conformation in solution at pH 2. The folding kinetics from molten globule to the native state was nearly identical to those from the unfolded state to the native state. This observation suggests that the equilibrium molten globule state of hydrophobic core variant ubiquitin is an on-pathway folding intermediate.     

Kinetics of hyaluronan hydrolysis in acidic solution at various pH values

Hyaluronic acid (HA) was hydrolyzed using varying temperatures (40, 60, and 80 degrees C) and acid concentrations (0.0010, 0.010, 0.10, 0.50, 1.0, and 2.0 M HCl). The degradation process was monitored by determination of weight average molecular weight ( M w) by size-exclusion chromatography with online multiangle laser light scattering, refractive index, and intrinsic viscosity detectors (SEC-MALLS-RI-visc) on samples taken out continuously during the hydrolysis. SEC-MALLS-RI-visc showed that the degradation gave narrow molecular weight distributions with polydispersity indexes ( M w/ M n) of 1.3-1.7. Kinetic plots of 1/ M w versus time gave linear plots showing that acid hydrolysis of HA is a random process and that it follows a first order kinetics. For hydrolysis in HCl at 60 and 80 degrees C, it was shown that the kinetic rate constant ( k h) for the degradation depended linearly on the acid concentration. Further, the dependence of temperature on the hydrolysis in 0.1 M HCl was found to give a linear Arrhenius plot (ln k h vs 1/ T), with an activation energy ( E a) of 137 kJ/mol and Arrhenius constant ( A) of 7.86 x 10 (15) h (-1). (1)H NMR spectroscopy was used to characterize the product of extensive hydrolysis (48 h at 60 degrees C in 0.1 M HCl). No indication of de- N-acetylation of the N-acetyl glucosamine (GlcNAc) units or other byproducts were seen. Additionally, a low molecular weight HA was hydrolyzed in 0.1 M DCl for 4 h at 80 degrees C. It was shown that it was primarily the beta-(1-->4)-linkage between GlcNAc and glucuronic acid (GlcA) that was cleaved during hydrolysis at pH < p K a,GlcA. The dependence of the hydrolysis rate constant was further studied as a function of pH between -0.3 and 5. The degradation was found to be random (linear kinetic plots) over the entire pH range studied. Further, the kinetic rate constant was found to depend linearly on pH in the region -0.3 to 3. Above this pH (around the p K a of HA), the kinetic constant decreased more slowly, probably due to either a change in polymer conformation or due to an increased affinity for protons due to the polymer becoming charged as the GlcA units dissociated.     

Conformational states of annexin VI in solution induced by acidic pH

Acidic pH-induced folding of annexin (Anx)VI in solution was investigated in order to study the mechanism of formation of ion channels by the protein in membranes. Using 2-(p-toluidino)naphthalene-6-sulfonic acid as a hydrophobic probe, it was demonstrated that AnxVI exerts a large change in hydrophobicity at acidic pH. Moreover, circular dichroism spectra indicated that the native state of AnxVI changes at acidic pH towards a state characterized by a significant loss of alpha-helix content and appearance of new beta-structures. These changes are reversible upon an increase of pH. It is postulated that the structural folding of AnxVI could explain how a soluble protein may undergo transition into a molecule able to penetrate the membrane hydrophobic region. The physiological significance of these observations is discussed.     

The diffusive Lotka-Volterra system with three species can have a stable non-constant equilibrium solution

Three examples of the diffusive 3-species Lotka-Volterra system with constant interaction parameters are given, and by bifurcation techniques shown to have stable spatially non-constant equilibrium solutions. One example is competitive; the second one predator-two-competing prey and the third involves two predators and a single prey.     

The effects of pH on hyaluronate as observed by light scattering

Hyaluronate was investigated over a wide pH range, and at near zero and intermediate ionic strength, using dynamic and total intensity light scattering. Commercially obtained rooster comb hyaluronate was purified, and solutions were prepared in pure water by low-power bath ultrasonication and subsequent filtering. These solutions were of low polydispersity and appeared to contain single molecules of hyaluronate. Despite the absence of added electrolyte, these solutions yielded well-behaved Zimm plots. Increasing ionic strength and changing pH decreased radii of gyration and increased diffusion constants. Except for what appeared to be slow hydrolysis at either extreme of pH, molecular weights remained constant under all pH and ionic strength conditions. Under all solvent conditions investigated, diffusion coefficients increased with decreasing hyaluronate concentration. Unsonicated, lightly centrifuged solutions without added electrolyte were polydisperse, and their light scattering intensity was dominated by what appeared to be stable hyaluronate aggregates. The results are interpreted in terms of the polyelectrolyte properties of hyaluronate and its tendency to form stable entanglements, especially at low ionic strength. Previous light scattering studies in the literature on hyaluronate have shown widely varying results. The present article briefly reviews this literature and attempts to explain the variation among the previous results, emphasizing the Kuhn statistical segment length as an indicator of whether results are influenced by polydispersity or contaminants causing hyaluronate aggregation.     

LexA repressor forms stable dimers in solution. The role of specific dna in tightening protein-protein interactions

Cooperativity in the interactions among proteins subunits and DNA is crucial for DNA recognition. LexA repressor was originally thought to bind DNA as a monomer, with cooperativity leading to tighter binding of the second monomer. The main support for this model was a high value of the dissociation constant for the LexA dimer (micromolar range). Here we show that the protein is a dimer at nanomolar concentrations under different conditions. The reversible dissociation of LexA dimer was investigated by the effects of hydrostatic pressure or urea, using fluorescence emission and polarization to monitor the dissociation process. The dissociation constant lies in the picomolar range (lower than 20 pM). LexA monomers associate with an unusual large volume change (340 ml/mol), indicating the burial of a large surface area upon dimerization. Whereas nonspecific DNA has no stabilizing effect, specific DNA induces tightening of the dimer and a 750-fold decrease in the K(d). In contrast to the previous model, a tight dimer rather than a monomer is the functional repressor. Accordingly, the LexA dimer only loses its ability to recognize a specific DNA sequence by RecA-induced autoproteolysis. Our work provides insights into the linkage between protein-protein interactions, DNA recognition, and DNA repair.     

The conformation of a B-DNA decamer is mainly determined by its sequence and not by crystal environment.

By comparing the conformations adopted by a double-stranded decameric B-DNA fragment in different crystal environments, we address the question of the degree of deformability of DNA helices. The three-dimensional structure of the self-complementary DNA decamer CCAGGCmeCTGG has been determined from crystals of space group P6 at 2.25 A resolution with an R value of 17.2% for 2407 1 sigma structure amplitudes. The oligonucleotide forms a B-type double helix with a characteristic sequence-dependent conformation closely resembling that of the corresponding unmethylated decamer, the structure of which is known from a high-resolution analysis of crystals of space group C2. Evidently, both the effects of single-site methylation and altered crystal environment on the DNA conformation are small. Therefore, double-helical DNA may possess sequence-determined conformational features that are less deformable than previously thought.     

Conformational change in a single molecular species, beta3, of beta-conglycinin in acidic ethanol solution

Several physicochemical experiments were done to obtain further information on the conformational changes occurring in beta-conglycinin in acidic-ethanol solution, using a single molecular species of this protein, beta3. By far-UV circular dichroism (CD), a transition from beta-sheet to alpha-helical structure was observed upon addition of acidic-ethanol, and the alpha-helix content was found to reach 76% in 70% ethanol (pH 2). From analyses of near-UV CD and difference absorption spectra, it was found that the tertiary structure of the beta3 species was significantly altered at ethanol concentrations between 10 and 20%. The profiles of binding of 1-anilinonaphthalene-8-sulfonic acid to the beta3 species during acidic-ethanol denaturation were indicative of the existence of intermediate conformers in the molten globule-like denaturation state. By measuring Fourier transform infrared spectra and estimating the Stokes radius by dynamic light scattering, the beta3 molecules were found to aggregate with an increase in ethanol concentration.     

Methanogenesis under acidic pH conditions in a semi-continuous reactor system

Operating an anaerobic digester at low pH could offer several advantages over operation at neutral pH. Most wastewater streams targeted for anaerobic digestion are inherently acidic, requiring alkalinity supplementation (at added expense) to buffer the pH at neutral. Additionally, previously published work completed by the authors using batch systems suggested that lowering the system pH could increase methane production by as much as 30%. The goal of this research was to evaluate the feasibility of sustaining methanogenesis at low pH in a semi-continuous laboratory-scale fermentor. Significant methane production was achieved in a system ranging in pH from approximately 4.0-5.3. Results show that, if the consortium is allowed to sufficiently acclimate to acidic conditions, methanogenesis can be maintained under acidic pH conditions, resulting in overall chemical oxygen demand (COD) reduction and methane production comparable to that achieved in a neutral pH system.     

Crystallographic characterization of two novel crystal forms of human insulin induced by chaotropic agents and a shift in pH

Background  

Insulin is a therapeutic protein that is widely used for the treatment of diabetes. Its biological function was discovered more than 80 years ago and it has since then been characterized extensively. Crystallization of the insulin molecule has always been a key activity since the protein is often administered by subcutaneous injections of crystalline insulin formulations. Over the years, insulin has been crystallized and characterized in a number of crystal systems.     

Fructose as a carbohydrate source yields stable pH and redox parameters in microcarrier cell culture

The pH and cytosolic NADH/NAD⁺ redox potential in microcarrier cultures of Madin-Darby canine kidney cells remain within physiological range when fructose is substituted for glucose in medium formulation. This difference is accounted for by the low rate of lactic acid production in cultures utilizing fructose as a primary carbohydrate source.     

Sequence specific conformation of a DNA decamer containing an adenine tract studied in solution by H-NMR spectroscopy

The decanucleotide duplex d(AAAACGTTTT)2 and a variety of phase-sensitive two-dimensional (2D) NMR experiments have been used to investigate the solution conformation of an adenine-tract and its junction with another DNA sequence. 2D nuclear Overhauser effect data confirm that the oligonucleotide has a general B-type DNA morphology but an array of unusual correlations implies that the adenine tract and the 5'-ApC junction have conformations more compatible with the modified X-ray structures recently reported for DNAs of similar sequence (Nelson, H.C.M., Finch, J.T., Luisi, B.F. and Klug, A. (1987) Nature 330, 221-226). The pattern and magnitude of interstrand NOEs from the adenine H2s to the sugar H1's of the complementary base to the 5'-neighbouring residue indicate that the A-T basepairs are highly propeller twisted and that the minor groove is narrowed, showing its greatest compression at the 3'-end of the tract at the 5'-ApC step. Quantifying spin-coupling interactions within the deoxyribose rings by analysing both 1D and high-resolution 2D DQF-COSY data reveals that the conformation of the purines is predominantly C2'-endo, with the pseudorotation phase angle P lying in the range 140-180 degrees. For the pyrimidines, however, there are distortions away from this standard B-type geometry with the data being best described by P values lying in the range 90-130 degrees (i.e., O4'-endo, C1'-exo). The sugar puckers of A1, T9 and T10 are dynamically distorted no doubt as a consequence of their positions at, or close to, the ends of the duplex. Thus the conformation of the adenine and thymine sugars within the oligo(dA) and oligo(dT) strands are different with an abrupt change in sugar puckering occurring at the 5'-ApC (5'-GpT) step. Peculiar chemical shifts values for A4H2, T7CH3 and sugar C5 H1', H2' and H2", together with a number of interresidue NOEs with unusual intensities, imply that there are also substantial modifications to basepair stacking interactions at this step. Taken as a whole, our data are consistent with the view that the conformational dislocation at the 5'-ApC dinucleotide results from a combination of slide and roll manoeuvres and that the junction between the AAAA and CG sequences is a potential nucleation site for DNA bending.     

A helical hairpin region of soluble annexin B12 refolds and forms a continuous transmembrane helix at mildly acidic pH

Annexins are soluble proteins that are best known for their ability to undergo reversible Ca(2+)-dependent binding to the surface of phospholipid bilayers. Recent studies, however, have shown that annexins also reversibly bind to membranes in a Ca(2+)-independent manner at mildly acidic pH. We investigated the structural changes that occur upon pH-dependent membrane binding by performing a nitroxide scan on the helical hairpin encompassing helices A and B in the fourth repeat of annexin B12. Residues 251-273 of annexin B12 were replaced, one at a time, with cysteine and then labeled with a nitroxide spin label. Electron paramagnetic resonance (EPR) mobility and accessibility analyses of soluble annexin B12 derivatives were in excellent agreement with the known crystal structure of annexin B12. However, EPR studies of annexin B12 derivatives bound to membranes at pH 4.0 indicated major structural changes in the scanned region. The helix-loop-helix structure present in the soluble protein was converted into a continuous transmembrane alpha-helix that was exposed to the hydrophobic core of the bilayer on one side and exposed to an aqueous pore on the other side. Asp-264 was on the hydrophobic membrane-exposed face of the amphipathic transmembrane helix, thereby suggesting that protonation of its carboxylate group stabilized the transmembrane form. Inspection of the amino acid sequence of annexin B12 revealed several other helical hairpin regions that might refold and form continuous amphipathic transmembrane helices in response to protonation of Asp or Glu switch residues on or near the hydrophobic face of the helix.     

Kinetics of pramlintide degradation in aqueous solution as a function of temperature and pH

The stability of the 37-amino acid peptide pramlintide, in aqueous solution, was studied as a function of pH and temperature. Samples of pramlintide formulated as a parenteral product were exposed to elevated temperatures and to realistic storage conditions for as long as 30 months. Pramlintide degradation was monitored by three high-performance liquid chromatography (HPLC) methods: a reversedphase (RP-HPLC) and a strong-cation exchange (SCX-HPLC) method for percentage purity determination by area normalization, plus a second RP-HPLC method for potency determinationversus external standards. The pH-rate profile for pramlintide shows increasing degradation rate constants with increasing pH over the range pH=3.5 to 5.0. The Arrhenius expression for pramlintide degradation at pH=4.0 over the temperature range 5°C to 50°C is In(k₀)=37.39−21.900/RT, where k₀ is the zero-order rate constant (in %/mo) for pramlintide degradation. The pramlintide parenteral product formulated at pH=4.0 is extremely stable, with percentage purity and percentage potency loss of only approximately 2% over 30 months at 5°C. The formulated pramlintide drug product has acceptable shelf life for long-term storage at 5°C and up to a 30-day patient use when stored at ambient temperature.     

Toxic phytoplankton as a keystone species in aquatic ecosystems: stable coexistence to biodiversity

The effect of allelochemicals released by toxic species in plankton community is often taken into account to reveal plankton biodiversity. Using a minimal chemostat model we show that the interaction between toxic and non‐toxic phytoplankton species with changing competitive effects among species due to allelopathy helps to promote the stable coexistence of many species on a single resource and hence can solve the paradox of plankton. We emphasize toxic phytoplankton as a keystone species that strongly uncovers its allelochemicals on other non‐toxic phytoplankton and enhances the species persistence and diversity in aquatic ecosystems. In addition, we analyze the consistency of ecosystem functioning and species diversity using a number of approaches, such as sampling hypothesis with selection and complementarity effects, cascading extinction–reinvasion, and examining system dynamics at different enrichment levels and toxicity. Our results suggest that chemostats with one toxic and one or more nontoxic phytoplankton species can be used for the experimental verification of the stable coexistence of many species on a single resource in aquatic ecology.     

The human mitochondrial Hsp60 in the APO conformation forms a stable tetradecameric complex

The human mitochondrial chaperonin is a macromolecular machine that catalyzes the proper folding of mitochondrial proteins and is of vital importance to all cells. This chaperonin is composed of 2 distinct proteins, Hsp60 and Hsp10, that assemble into large oligomeric complexes that mediate the folding of non-native polypeptides in an ATP dependent manner. Here, we report the bacterial expression and purification of fully assembled human Hsp60 and Hsp10 recombinant proteins and that Hsp60 forms a stable tetradecameric double-ring conformation in the absence of co-chaperonin and nucleotide. Evidence of the stable double-ring conformation is illustrated by the 15 Å resolution electron microscopy reconstruction presented here. Furthermore, our biochemical analyses reveal that the presence of a non-native substrate initiates ATP-hydrolysis within the Hsp60/10 chaperonin to commence protein folding. Collectively, these data provide insight into the architecture of the intermediates used by the human mitochondrial chaperonin along its protein folding pathway and lay a foundation for subsequent high resolution structural investigations into the conformational changes of the mitochondrial chaperonin.     

Crystal structure of reduced and of oxidized peroxiredoxin IV enzyme reveals a stable oxidized decamer and a non-disulfide-bonded intermediate in the catalytic cycle

Peroxiredoxin IV (PrxIV) is an endoplasmic reticulum-localized enzyme that metabolizes the hydrogen peroxide produced by endoplasmic reticulum oxidase 1 (Ero1). It has been shown to play a role in de novo disulfide formation, oxidizing members of the protein disulfide isomerase family of enzymes, and is a member of the typical 2-Cys peroxiredoxin family. We have determined the crystal structure of both reduced and disulfide-bonded, as well as a resolving cysteine mutant of human PrxIV. We show that PrxIV has a similar structure to other typical 2-Cys peroxiredoxins and undergoes a conformational change from a fully folded to a locally unfolded form following the formation of a disulfide between the peroxidatic and resolving cysteine residues. Unlike other mammalian typical 2-Cys peroxiredoxins, we show that human PrxIV forms a stable decameric structure even in its disulfide-bonded state. In addition, the structure of a resolving cysteine mutant reveals an intermediate in the reaction cycle that adopts the locally unfolded conformation. Interestingly the peroxidatic cysteine in the crystal structure is sulfenylated rather than sulfinylated or sulfonylated. In addition, the peroxidatic cysteine in the resolving cysteine mutant is resistant to hyper-oxidation following incubation with high concentrations of hydrogen peroxide. These results highlight some unique properties of PrxIV and suggest that the equilibrium between the fully folded and locally unfolded forms favors the locally unfolded conformation upon sulfenylation of the peroxidatic cysteine residue.