Discrete roles of copper ions in chemical unfolding of human ceruloplasmin

Human ceruloplasmin (CP) is a multicopper oxidase essential for normal iron homeostasis. The protein has six beta-barrel domains with one type 1 copper in each of domains 2, 4, and 6; the remaining copper ions form a catalytic trinuclear cluster, one type 2 and two type 3 coppers, at the interface between domains 1 and 6. We have characterized urea-induced unfolding of holo- and apo-forms of CP by far-UV circular dichroism, intrinsic fluorescence, 8-anilinonaphthalene-1-sulfonic acid binding, visible absorption, copper content, and oxidase activity probes (pH 7, 23 degrees C). We find that holo-CP unfolds in a complex reaction with at least one intermediate. The formation of the intermediate correlates with decreased secondary structure, exposure of aromatics, loss of two coppers, and reduced oxidase activity; this step is reversible, indicating that the trinuclear cluster remains intact. Further additions of urea trigger complete protein unfolding and loss of all coppers. Attempts to refold this species result in an inactive apoprotein with molten-globule characteristics. The apo-form of CP also unfolds in a multistep reaction, albeit the intermediate appears at a slightly lower urea concentration. Again, correct refolding is possible from the intermediate but not the unfolded state. Our study demonstrates that in vitro equilibrium unfolding of CP involves intermediates and that the copper ions are removed in stages. When the catalytic site is finally destroyed, refolding is not possible at neutral pH. This implies a mechanistic role for the trinuclear metal cluster as a nucleation point, aligning domains 1 and 6, during CP folding in vivo.     

Synthesis, structure and redox properties of an unexpected trinuclear copper(II) complex with aspartame: [Cu(apm)2Cu(μ-N,O:O′-apm)2(H2O)Cu(apm)2(H2O)] · 5H2O

Structural, magnetic and spectroscopic data of a new trinuclear copper(II) complex with the ligand aspartame (apm) are described. [Cu(apm)₂Cu(μ-N,O:O′-apm)₂(H₂O)Cu(apm)₂(H₂O)] · 5H₂O crystallizes in the triclinic system, space group P1 (#1) with a = 7.3300(1) Å, b = 15.6840(1) Å, c = 21.5280(1) Å, α = 93.02(1)°, β = 93.21(1)°, γ = 92.66(1)° and Z = 1. Aspartame coordinates to Cu(II) through the carboxylate and β-amino groups. The carboxylate groups of the two central ligands act as bidentate bridges in a syn-anti conformation while the carboxylate groups of the four peripheral ligands are monodentate in a syn conformation. The central copper ion is in a distorted square pyramidal geometry with the apical position being occupied by one oxygen atom of the water molecule. The two terminal copper(II) atoms are coordinated to the ligands in the same position but their coordination sphere differs from each other due to the fact that one copper atom has a water molecule in an apical position leading to an octahedral coordination sphere while the other copper atom is exclusively coordinated to aspartame ligands forming a distorted square pyramidal coordination sphere. Thermal analysis is consistent with the X-ray structure. EPR spectra and CV curves indicate a rupture of the trinuclear framework when this complex is dissolved in ethanol or DMF, forming a mononuclear species, with a tetragonal structure.     

Reversible heat inactivation of copper sites precedes thermal unfolding of molluscan (Rapana thomasiana) hemocyanin

Hemocyanin (Hc) is a type-3 copper protein, containing dioxygen-binding active sites consisting of paired copper atoms. In the present study the thermal unfolding of the Hc from the marine mollusc Rapana thomasiana (RtH) has been investigated by combining differential scanning calorimetry, Fourier transform infrared (FTIR) and UV–vis absorption spectroscopy. Two important stages in the unfolding pathway of the Hc molecule were discerned. A first event, with nonmeasurable heat absorption, occurring around 60 °C, lowers the binding of dioxygen to the type-3 copper groups. This pretransition is reversible and is ascribed to a slight change in the tertiary structure. In a second stage, with midpoint around 80 °C, the protein irreversibly unfolds with a loss of secondary structure and formation of amorphous aggregates. Experiments with the monomeric structural subunits, RtH1 and RtH2, indicated that the heterogeneity in the process of thermal denaturation can be attributed to the presence of multiple 50 kDa functional units with different stability. In accordance, the irreversible unfolding of a purified functional unit (RtH2-e) occurred at a single transition temperature. At slightly alkaline pH (Tris buffer) the C-terminal β-sheet rich domain of the functional unit starts to unfold before the α-helix-rich N-terminal (copper containing) domain, triggering the collapse of the global protein structure. Even around 90 °C some secondary structure is preserved as shown by the FTIR spectra of all investigated samples, confirming the high thermostability of molluscan Hc.     

Rapid thermal responses and thermal tolerance in adult codling moth Cydia pomonella (Lepidoptera: Tortricidae)

In order to preserve key activities or improve survival, insects facing variable and unfavourable thermal environments may employ physiological adjustments on a daily basis. Here, we investigate the survival of laboratory-reared adult Cydia pomonella at high or low temperatures and their responses to pre-treatments at sub-lethal temperatures over short time-scales. We also determined critical thermal limits (CTLs) of activity of C. pomonella and the effect of different rates of cooling or heating on CTLs to complement the survival assays. Temperature and duration of exposure significantly affected adult C. pomonella survival with more extreme temperatures and/or longer durations proving to be more lethal. Lethal temperatures, explored between −20 °C to −5 °C and 32 °C to 47 °C over 0.5, 1, 2, 3 and 4 h exposures, for 50% of the population of adult C. pomonella were −12 °C for 2 h and 44 °C for 2 h. Investigation of rapid thermal responses (i.e. hardening) found limited low temperature responses but more pronounced high temperature responses. For example, C. pomonella pre-treated for 2 h at 5 °C improved survival at −9 °C for 2 h from 50% to 90% (p < 0.001). At high temperatures, pre-treatment at 37 °C for 1 h markedly improved survival at 43 °C for 2 h from 20% to 90% (p < 0.0001). We also examined cross-tolerance of thermal stressors. Here, low temperature pre-treatments did not improve high temperature survival, while high temperature pre-treatment (37 °C for 1 h) significantly improved low temperature survival (−9 °C for 2 h). Inducible cross-tolerance implicates a heat shock protein response. Critical thermal minima (CTmin) were not significantly affected by cooling at rates of 0.06, 0.12 and 0.25 °C min⁻¹ (CTmin range: 0.3-1.3 °C). By contrast, critical thermal maxima (CTmax) were significantly affected by heating at these rates and ranged from 42.5 to 44.9 °C. In sum, these results suggest pronounced plasticity of acute high temperature tolerance in adult C. pomonella, but limited acute low temperature responses. We discuss these results in the context of local agroecosystem microclimate recordings. These responses are significant to pest control programmes presently underway and have implications for understanding the evolution of thermal tolerance in these and other insects.     

Thermal responses of juvenile squaretail mullet (Liza vaigiensis) and juvenile crescent terapon (Terapon jarbua) acclimated at near-lethal temperatures, and the implications for climate change

The negative effects of climate alteration on coral reef fishes receive ever increasing attention; however, implications of rising sea temperatures on fishes inhabiting marine nursery environments are poorly understood. We used critical thermal methodology to quantify critical thermal maxima (CTmaxima) of juvenile squaretail mullet (Liza vaigiensis) and juvenile crescent terapon (Terapon jarbua) captured from shallow seagrass nursery areas around Hoga Island, southeast Sulawesi, Indonesia. We tested the hypothesis that these distantly related fishes, when acclimated to cycling temperatures, would display higher CTmaxima than groups acclimated at constant temperatures. Groups of mullet acclimated to a constant temperature of 37 °C and temperature cycles of 35 to 39 °C or 37 to 41 °C displayed statistically similar mean CTmaxima of 44.7, 44.4 and 44.8 °C, respectively. Likewise, terapon acclimated at temperature cycles of 37 to 40 °C did not display a higher CTmaxima than fish acclimated at a constant temperature of 37 °C, with both acclimation groups' mean CTmaxima equal to 43.8 °C. Acclimation to higher cycling temperatures did not result in significant upper temperature tolerance acquisition for either species; however, mullet values were significantly higher than those seen in terapon (P < 0.0001). These data suggest that mullet and terapon will not suffer direct thermal effects should shallow nursery temperature increases be marginally higher than 1-2 °C above ~ 27 °C, and they provide evidence that the upper thermal tolerance of fishes inhabiting shallow seagrass and mangrove areas can approach the biokinetic limits for vertebrate life. Tropical marine fishes inhabiting fringing nursery environments may have the upper thermal tolerance necessary to endure substantial increases in sea temperatures.     

Synthesis, crystal structure and magnetism of centrosymmetric linear trinuclear copper(II) complex of pyridine nitronyl nitroxide derivative

Novel stable dark-green solid adducts (2) were isolated from the reaction of 2-(4,4,5,5-tetramethyl-3-oxylimidazoline-1-oxide)-5-bromopyridine (1) with copper(II)-hexafluoroacetylacetonate hydrate (Cu(hfac)₂ · xH₂O), namely [1₂ • {Cu(hfac)₂}₃]. The X-ray structure revealed that, the complex is linear and centrosymmetric in the triclinic space group. The striking feature of the complex is, the presence of two types of copper coordination geometries with the radical 1, a distorted octahedron at the center and two terminal octahedron structures, where the copper and the nitronyl nitroxide oxygen binding is stronger in the latter case due to “chelation effect”. The central copper is surrounded by O₆ donor sets and the terminal coppers are surrounded by N,O₅ donor sets. The three copper ions in the complex are collinear with shortest intramolecular metal-metal distance ca. 7.75 Å. The angles between Cu-O-N also differ considerably due to the different overlapping modes of nitronyl nitroxide oxygen with copper, for chelating type it is 111.25° and for the non-chelating one it is 142.85°. Two exchange couplings are possible for this kind of system. The magnetic behavior shows that the coupling is antiferromagnetic between the radical and the terminal coppers, with J₁=−440 cm⁻¹ and weakly ferromagnetic between the central copper and the neighboring radicals with J₂=10 cm⁻¹.     

Angular trinuclear copper(II) complexes of N4O-donor ligand: Syntheses, crystal structures and magnetic properties

Two trinuclear copper(II) complexes of a Schiff-base type N₄O-donor ligand (LH) derived from 4,4,9,9-tetramethyl-5,8-diazadodecane-2,11-dione and 1,3-diaminopropan-2-ol are reported. Complex [Cu₃L₂(ClO₄)₄] (1) has an angular C₂-symmetric trinuclear core as revealed from single-crystal X-ray diffraction studies. The terminal coppers are in square-pyramidal geometry with an N₃O₂ coordination environment while the central one is in octahedral geometry with an N₂O₄ donor environment. Complex [Cu₃L₂(ClO₄)(N₃)(H₂O)](ClO₄)₂ · H₂O (2) has an unsymmetrical trinuclear core with an intramolecular hydrogen bonding interaction between the water and azide anion coordinated to Cu(1) and Cu(3) center, respectively. All the copper centers in 2 are in square-pyramidal geometry. The average Cu?Cu distance between closest metal ions in both the complexes is 3.897 Å. The coordination environment of coppers in 1 approximately mimics that of multicopper oxidases in the oxidized form and the environment in 2 mimics that of the azide derivative of ascorbate oxidase. Both 1 and 2 exhibit doublet spin ground state due to strong antiferromagnetic coupling operating through the alkoxo-bridged oxygen atoms between the copper centers.     

Interrupted Catalysis: The EF4 (LepA) Effect on Back-Translocation

EF4, although structurally similar to the translocase EF-G, promotes back-translocation of tRNAs on the ribosome and is important for bacterial growth under certain conditions. Here, using a coordinated set of in vitro kinetic measures, including changes in the puromycin reactivity of peptidyl-tRNA and in the fluorescence of labeled tRNAs and mRNA, we elucidate the kinetic mechanism of EF4-catalyzed back-translocation and determine the effects of the translocation inhibitors spectinomycin and viomycin on the process. EF4-dependent back-translocation proceeds from a post-translocation (POST) complex to a pre-translocation (PRE) complex via a four-step kinetic scheme (i.e., POST → I₁ → I₂ → I₃ → PRE, which is not the simple reverse of translocation). During back-translocation, movements of the tRNA core regions and of mRNA are closely coupled to one another but are sometimes decoupled from movement of the 3′-end of peptidyl-tRNA. EF4 may be thought of as performing an interrupted catalysis of back-translocation, stopping at the formation of I₃ rather than catalyzing the complete process of back-translocation culminating in PRE complex formation. The delay in polypeptide elongation resulting from transient accumulation of I₃ is likely to be important for optimizing functional protein biosynthesis.     

An endo-(1→3)-β-d-glucanase from the scallop Chlamys albidus: catalytic properties, cDNA cloning and secondary-structure characterization

An endo-(1→3)-β-d-glucanase (L₀) with molecular mass of 37 kDa was purified to homogeneity from the crystalline style of the scallop Chlamys albidus. The endo-(1→3)-β-d-glucanase was extremely thermolabile with a half-life of 10 min at 37 °C. L₀ hydrolyzed laminaran with K_m ∼ 0.75 mg/mL, and catalyzed effectively transglycosylation reactions with laminaran as donor and p-nitrophenyl β d-glucoside as acceptor (K_m ∼ 2 mg/mL for laminaran) and laminaran as donor and as acceptor (K_m ∼ 5 mg/mL) yielding p-nitrophenyl β d-glucooligosaccharides (n = 2-6) and high-molecular branching (1→3),(1→6)-β-d-glucans, respectively. Efficiency of hydrolysis and transglycosylation processes depended on the substrate structure and decreased appreciably with the increase of the percentage of β-(1→6)-glycosidic bonds, and laminaran with 10% of β-(1→6)-glycosidic bonds was the optimal substrate for both reactions. The CD spectrum of L₀ was characteristic for a protein with prevailing β secondary-structural elements. Binding L₀ with d-glucose as the best acceptor for transglycosylation was investigated by the methods of intrinsic tryptophan fluorescence and CD. Glucose in concentration sufficient to saturate the enzyme binding sites resulted in a red shift in the maximum of fluorescence emission of 1-1.5 nm and quenching the Trp fluorescence up to 50%. An apparent association constant of L₀ with glucose (K_a = 7.4 × 10⁵ ± 1.1 × 10⁵ M⁻¹) and stoichiometry (n = 13.3 ± 0.7) was calculated. The cDNA encoding L₀ was sequenced, and the enzyme was classified in glycoside hydrolases family 16 on the basis of the amino acid sequence similarity.     

Synthesis, crystal structure, magnetic properties and electrochemical behaviour of the mixed valence compound [Cu(CN)3Cu(C3H10N2)2]

The binuclear mixed valence copper(I/II) compound [Cu^I(CN)₃Cu^II(tn)₂] (1) (tn = propane-1,3-diamine) and its acetonitrile adduct [Cu^I(CN)₃Cu^II(tn)₂] · 2MeCN (2) have been synthesized. Complex 1 crystallizes triclinic, space group , a = 8.117(2) Å, b = 8.389(2) Å, c = 11.920(2) Å, α = 108.728(3)°, β = 100.024(3)°, γ = 104.888(4)°, Z = 2, and compound 2 monoclinic, space group P2₁/m, a = 8.752(2) Å, b = 13.243(3) Å, c = 9.549(2) Å, β = 114.678(4)°, Z = 2. In both crystal structures, the binuclear [Cu^I(CN)₃Cu^II(tn)₂] complex with slightly different bonding geometries is formed. One of the three nitrogen atoms of a Cu^I(CN)₃ moiety is coordinated to Cu(II) at the apex of a square-pyramid with two chelating ligands tn on its base. The shortest intramolecular Cu^II?Cu^II distance in 1 is 5.640(7) Å. The EPR behaviour of 1 has been investigated at room temperature and at 77 K. The magnetic properties were measured in the temperature range 1.8-300 K.     

Purification and characterization of a thermostable α-amylase produced by the fungus Paecilomyces variotii

An α-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The α-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pI value of 4.5. Temperature and pH optima were 60 °C and 4.0, respectively. The enzyme was stable for 1 h at 55 °C, showing a t₅₀ of 53 min at 60 °C. Starch protected the enzyme against thermal inactivation. The α-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K_m of α-amylase on Reagen® and Sigma® starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to α-amylases from Bacillus sp. These results confirmed that the studied enzyme was an α-amylase ((1→4)-α-glucan glucanohydrolase).     

A cryoprotective and cold-adapted 1,3-β-endoglucanase from cherimoya (Annona cherimola) fruit

A 1,3-β-glucanase with potent cryoprotective activity was purified to homogeneity from the mesocarp of CO₂-treated cherimoya fruit (Annona cherimola Mill.) stored at low temperature using anion exchange and chromatofocusing chromatography. This protein was characterized as a glycosylated endo-1,3-β-glucanase with a M_r of 22.07 kDa and a pI of 5.25. The hydrolase was active and stable in a broad acidic pH range and it exhibited maximum activity at pH 5.0. It had a low optimum temperature of 35 °C and it retained 40% maximum activity at 5 °C. The purified 1,3-β-glucanase was relatively heat unstable and its activity declined progressively at temperatures above 50 °C. Kinetic studies revealed low k_cat (3.10 ± 0.04 s⁻¹) and K_m (0.32 ± 0.03 mg ml⁻¹) values, reflecting the intermediate efficiency of the protein in hydrolyzing laminarin. Moreover, a thermodynamic characterization revealed that the purified enzyme displayed a high k_cat at both 37 and 5 °C, and a low E_a (6.99 kJ mol⁻¹) within this range of temperatures. In vitro functional studies indicated that the purified 1,3-β-glucanase had no inhibitory effects on Botrytis cinerea hyphal growth and no antifreeze activity, as determined by thermal hysteresis analysis using differential scanning calorimetry. However, a strong cryoprotective activity was observed against freeze-thaw inactivation of lactate dehydrogenase. Indeed, the PD₅₀ was 8.7 μg ml⁻¹ (394 nM), 9.2-fold higher (3.1 on a molar basis) than that of the cryoprotective protein BSA. Together with the observed accumulation of glycine-betaine in CO₂-treated cherimoya tissues, these results suggest that 1,3-β-glucanase could be functionally implicated in low temperature-defense mechanism activated by CO₂.     

Optical properties of Ag(tripod)X with tripod = 1,1,1-tris(diphenyl-phosphinomethyl)ethane and X = Cl and I: Intraligand and ligand-to-ligand charge transfer

The complexes Ag^I(tripod)X with tripod = 1,1,1-tris(diphenylphosphinomethyl)ethane and X⁻ = Cl⁻ and I⁻ are luminescent in solution at r.t. It is suggested that the emission is a phosphorescence which originates from a tripod intraligand state for X = Cl (λ_max = 464 nm) and a X⁻ → tripod ligand-to-ligand charge transfer state for X = I (λ_max = 482 nm).     

Synthesis and luminescent properties of gadolinium aluminates phosphors

In this work, aluminum-gadolinium oxides with different phases were prepared by the non-hydrolytic sol-gel route, using lower temperatures than those employed in methods such as solid-state reaction and Pechini method. The influence of heating treatment on sample structure was investigated. The formation process and the local structure of the samples are discussed on the basis of thermal, X-ray diffraction, photoluminescence (PL) spectroscopy, and infrared spectroscopy analyses. The quantum efficiency of Eu³⁺ in the different phases obtained in this studied was evaluated. Initial crystallization and the GdAlO₃ phase were observed at temperatures around 400 °C. PL data of all the samples revealed the characteristic transition bands arising from the ⁵D₀ → ⁵F_J (J = 0, 1, 2, 3, and 4) manifolds under maximum excitation at 275, 393, and 467 nm in all cases. The ⁵D₀ → ⁷F₂ transition often dominates the emission spectra, indicating that the Eu³⁺ ion occupies a site without inversion center.     

Gene cloning and catalysis features of a new mannitol-1-phosphate dehydrogenase (BbMPD) from Beauveria bassiana

A long-chain mannitol-1-phosphate dehydrogenase (MPD) was characterized for the first time from fungal entomopathogen Beauveria bassiana by gene cloning, heterogeneous expression and activity analysis. The cloned gene BbMPD consisted of a 1334-bp open reading frame (ORF) with a 158-bp intron and the 935-bp upstream and 780-bp downstream regions. The ORF-encoded 391-aa protein (42 kDa) showed less than 75% sequence identity to 17 fungal MPDs documented and shared two conserved domains with the fungal MPD family at the N- and C-terminus, respectively. The new enzyme was expressed well in the Luria-Bertani culture of engineered Escherichia coli BL21 by 16-h induction of 0.5 mM isopropyl 1-thio-β-d-galactopyranoside at 20 °C after 5-h growth at 37 °C. The purified BbMPD exhibited a high catalytic efficiency (k_cat/K_m) of 1.31 × 10⁴ mM⁻¹ s⁻¹ in the reduction of the highly specific substrate d-fructose-6-phosphate to d-mannitol-1-phosphate. Its activity was maximal at the reaction regime of 37 °C and pH 7.0 and was much more sensitive to Cu²⁺ and Zn²⁺ than to Li⁺ and Mn²⁺. The results indicate a crucial role of BbMPD in the mannitol biosynthesis of B. bassiana.     

Heat tolerance, behavioural temperature selection and temperature-dependent respiration in larval Octopus huttoni

This study reports temperature effects on paralarvae from a benthic octopus species, Octopus huttoni, found throughout New Zealand and temperate Australia. We quantified the thermal tolerance, thermal preference and temperature-dependent respiration rates in 1-5 days old paralarvae. Thermal stress (1 °C increase h⁻¹) and thermal selection (∼10-24 °C vertical gradient) experiments were conducted with paralarvae reared for 4 days at 16 °C. In addition, measurement of oxygen consumption at 10, 15, 20 and 25 °C was made for paralarvae aged 1, 4 and 5 days using microrespirometry. Onset of spasms, rigour (CT_max) and mortality (upper lethal limit) occurred for 50% of experimental animals at, respectively, 26.0±0.2 °C, 27.8±0.2 °C and 31.4±0.1 °C. The upper, 23.1±0.2 °C, and lower, 15.0±1.7 °C, temperatures actively avoided by paralarvae correspond with the temperature range over which normal behaviours were observed in the thermal stress experiments. Over the temperature range of 10 °C-25 °C, respiration rates, standardized for an individual larva, increased with age, from 54.0 to 165.2 nmol larvae⁻¹ h⁻¹ in one-day old larvae to 40.1-99.4 nmol h⁻¹ at five days. Older larvae showed a lesser response to increased temperature: the effect of increasing temperature from 20 to 25 °C (Q₁₀) on 5 days old larvae (Q₁₀=1.35) was lower when compared with the 1 day old larvae (Q₁₀=1.68). The lower Q₁₀ in older larvae may reflect age-related changes in metabolic processes or a greater scope of older larvae to respond to thermal stress such as by reducing activity. Collectively, our data indicate that temperatures >25 °C may be a critical temperature. Further studies on the population-level variation in thermal tolerance in this species are warranted to predict how continued increases in ocean temperature will limit O. huttoni at early larval stages across the range of this species.     

A new ferromagnetically coupled μ-alkoxo-μ-acetato copper(II) trinuclear complex: [Cu3(H2tea)(Htea)(CH3COO)2](ClO4) (H3tea = triethanolamine)

A μ-alkoxo-μ-acetato trinuclear copper(II) complex, [Cu₃(H₂tea)(Htea)(CH₃COO)₂](ClO₄) 1, has been synthesized by reacting copper(II) perchlorate, triethanolamine and sodium acetate. The unit cell contains two centrosymmetric, crystallographically independent trinuclear Cu(II) complexes and two ions. The crystallographically independent trinuclear Cu(II) complexes differ mainly in some of their geometry parameters. The coordination environment of the central copper atom is square-planar, in one trinuclear entity, and elongated octahedral in the other one (in this last case, the coordination number of the central copper atom increases through the semicoordination of an oxygen atom arising from the aminoalcohol). The acetato groups exhibit the classical syn-syn bridging mode. The distances between the copper(II) ions in the two entities are, respectively: 3.043(3) and 3.034(4) Å. The cryomagnetic investigation of 1 reveals a ferromagnetic interaction between the copper(II) ions (J = +84 cm⁻¹), which is due to a countercomplementary effect of the acetato and alkoxo bridges.     

Thermodynamically stable aggregation-resistant antibody domains through directed evolution

Protein aggregates are usually formed by interactions between unfolded or partially unfolded species, and often occur when a protein is denatured by, for example, heat or low pH. In earlier work, we used a Darwinian selection strategy to create human antibody variable domains that resisted heat aggregation. The repertoires of domains were displayed on filamentous phage and denatured (at 80 °C in pH 7.4), and folded domains were selected by binding to a generic ligand after cooling. This process appeared to select for domains with denatured states that resisted aggregation, but the domains only had low free energies of folding (ΔG_N-D^o = 15-20 kJ/mol at 25 °C in pH 7.4). Here, using the same phage repertoire, we have extended the method to the selection of domains resistant to acid aggregation. In this case, however, the thermodynamic stabilities of selected domains were higher than those selected by thermal denaturation (under both neutral and acidic conditions; ΔG_N-D^o = 26-47 kJ/mol at 25 °C in pH 7.4, or ΔG_N-D^o = 27-34 kJ/mol in pH 3.2). Furthermore, we identified a key determinant (Arg28) that increased the aggregation resistance of the denatured states of the domains at low pH without compromising their thermodynamic stabilities. Thus, the selection process yielded domains that combined thermodynamic stability and aggregation-resistant unfolded states. We suggest that changes to these properties are controlled by the extent to which the folding equilibrium is displaced during the process of selection.     

Thermal preference and tolerance of Megastrea (Lithopoma) undosa (Wood, 1828; Gastropoda: Turbinidae)

The thermoregulatory behavior of the wavy turban snail Megastrea (Lithopoma) undosa was determined in a horizontal thermal gradient and was 16.31 in day cycle and 14.4 °C in night cycle. Displacement velocity of adults was 29.3±4.2 cm h⁻¹ during the light phase and 26.1±3.2 cm h⁻¹ during the dark phase. The critical thermal maxima of the wavy turban snail were determined. As a measure of thermal tolerance, snails were subjected to increasing water temperatures at a rate of 1 °C every 30 min until they were detached from the substrate. The critical thermal maximum at 50% was 29.7 °C.