Structural nonequivalence of the alpha- and beta- heme-pockets in human methemoglobin. A proton magnetic relaxation study in solution.

Solvent-proton longitudinal magnetic relaxation rates as dependent on temperature were measured for human (H)/canine (C) valency hybrids of the type {α^H(III)β^C(II)}₂ and {α^C(II)β^H(III)}₂. The two metheme irons in the human methemoglobin chains induce quite different proton magnetic relaxation (pmr) rates reflecting a tighter β-heme-pocket compared to the α subunit. Both heme-pockets appear to be loosened in the presence of inositol hexaphosphate (IHP) although this allosteric effector binds only to the β chains, the binding assumed to be the same for canine as for human hemoglobin. The subunit nonequivalence is retained also in the T-quaternary state induced by IHP. In the species hybrids the pmr rates due to the metheme iron are sensitive to the valency (ligand) state, which was either CO or H₂O in the partner half of the hybrid. All results show very clearly the interrelationship of the tertiary (protomer) structure with the quaternary (oligomer) structure in hemoglobin.     

Unique assignment of inter-subunit association in GABAA α1β3γ2 receptors determined by molecular modeling

Recent publications defined requirements for inter-subunit contacts in a benzodiazepine-sensitive GABA_A receptor (GABA_ARα1β3γ2). There is strong evidence that the heteropentameric receptor contains two α1, two β3, and one γ2 subunit. However, the available data do not distinguish two possibilities: When viewed clockwise from an extracellular viewpoint the subunits could be arranged in either γ2β3α1β3α1 or γ2α1β3α1β3 configurations. Here we use molecular modeling to thread the relevant GABA_AR subunit sequences onto a template of homopentameric subunits in the crystal structure of the acetylcholine binding protein (AChBP). The GABA_A sequences are known to have 15-18% identity with the acetylcholine binding protein and nearly all residues that are conserved within the nAChR family are present in AChBP. The correctly aligned GABA_A sequences were threaded onto the AChBP template in the γ2β3α1β3α1 or γ2α1β3α1β3  arrangements. Only the γ2α1β3α1β3 arrangement satisfied three known criteria: (1) α1 His¹⁰² binds at the γ2 subunit interface in proximity to γ2 residues Thr¹⁴², Phe⁷⁷, and Met¹³⁰; (2) α1 residues 80-100 bind near γ2 residues 91-104; and (3) α1 residues 58-67 bind near the β3 subunit interface. In addition to predicting the most likely inter-subunit arrangement, the model predicts which residues form the GABA and benzodiazepine binding sites.     

The molecular mechanism for human metallothionein-3 to protect against the neuronal cytotoxicity of Aβ1–42 with Cu ions

Aggregation and cytotoxicity of Aβ with redox-active metals in neuronal cells have been implicated in the progression of Alzheimer disease. Human metallothionein (MT) 3 is highly expressed in the normal human brain and is downregulated in Alzheimer disease. Zn₇MT3 can protect against the neuronal toxicity of Aβ by preventing copper-mediated Aβ aggregation, abolishing the production of reactive oxygen species (ROS) and the related cellular toxicity. In this study, we intended to decipher the roles of single-domain proteins (α/β) and the α–β domain–domain interaction of Zn₇MT3 to determine the molecular mechanism for protection against the neuronal cytotoxicity of Aβ_1–42 with copper ions. With this in mind, the α and β single-domain proteins, heterozygous β(MT3)–α(MT1), and a linker-truncated mutant ?31–34 were prepared and characterized. In the presence/absence of various Zn₇MT3 proteins, the Aβ_1–42–Cu²⁺-mediated aggregation, the production of ROS, and the cellular toxicity were investigated by transmission electron microscopy, ROS assay by means of a fluorescent probe, and SH-SY5Y cell viability, respectively. The β domain cannot abolish Aβ_1–42–Cu²⁺-induced aggregation, and neither the β domain nor the α domain can quench the production of ROS because of the redox cycling of Aβ–Cu²⁺. Similarly to wild-type Zn₇MT3, the heterozygous β(MT3)–α(MT1) possesses the characteristic of alleviating Aβ_1–42 aggregation and oxidative stress to neuronal cells. Therefore, the two domains through the linker Lys-Lys-Ser form a cooperative unit, and each of them is indispensable in conducting its bioactivity. The α domain plays an important role in modulating the stability of the metal–thiolate cluster, and the α–β domain–domain interaction through the linker is critical for its protective role in the brain.     

Mineralocorticoids modulate the expression of the β-3 subunit of the Na+, K+-ATPase in the renal collecting duct

Renal sodium reabsorption depends on the activity of the Na⁺,K⁺-ATPase α/β heterodimer. Four α (α_1–4) and 3 β (β_1–3) subunit isoforms have been described. It is accepted that renal tubule cells express α₁/β₁ dimers. Aldosterone stimulates Na⁺,K⁺-ATPase activity and may modulate α₁/β₁ expression. However, some studies suggest the presence of β₃ in the kidney. We hypothesized that the β₃ isoform of the Na⁺,K⁺-ATPase is expressed in tubular cells of the distal nephron, and modulated by mineralocorticoids. We found that β₃ is highly expressed in collecting duct of rodents, and that mineralocorticoids decreased the expression of β₃. Thus, we describe a novel molecular mechanism of sodium pump modulation that may contribute to the effects of mineralocorticoids on sodium reabsorption.     

Effect of thymosin peptides on the chick chorioallantoic membrane angiogenesis model

The effect of α- and β-thymosin peptides, namely prothymosin α (ProTα), thymosin α₁ (Tα1), parathymosin α (ParaTα), thymosin β₄ (Tβ4), thymosin β₁₀ (Tβ10), and thymosin β₉ (Tβ9), on the angiogenesis process was investigated using the chick chorioallantoic membrane as an in vivo angiogenesis model. The thymosin peptides tested were applied in 10 μl aliquots containing 0.01–4 nmoles of Tβ4, Tβ10 or Tβ9, 0.016–6.66 nmoles of Tα1, 4.1 pmoles–1.66 nmoles of ProTα, and 4.4 pmoles–1.76 nmoles of ParaTα. Phorbol 12-myristate 13-acetate and hydrocortisone were also used as positive and negative control, respectively. Tβ4, ProTα and Tα1 were found to enhance angiogenesis, while Tβ10, Tβ9 and ParaTα exhibited an inhibitory effect on the angiogenesis process. When mixtures of Tβ4 and Tβ10 containing active amounts of the two peptides at different proportions were applied, the promoting effect of Tβ4 on angiogenesis was reversed in the presence of increasing concentrations of Tβ10 and vice versa. The effect of Tβ10, Tβ9, ProTα and ParaTα, in parallel with Tβ4 and Tα1, on the angiogenesis process was investigated for the first time as far as we know and the results of this study offer more insight into the biological regulatory roles of thymosin peptides, and provide helpful information about their therapeutic potential. Whether these agents could be used either as inhibitors of angiogenesis in disease states where uncontrolled angiogenesis is involved, e.g. in carcinogenesis, or as angiogenesis promoters that could be useful in wound healing, fracture repair, peptic ulcers etc., remains to be further studied.     

Dimeric antioxidant and cytotoxic triterpenoid saponins from Terminalia ivorensis A. Chev.

Three saponins, including two dimeric triterpenoid glucosides possessing an unusual skeleton, ivorenosides A and B, and a monomeric triterpenoid saponin (ivorenoside C), together with the known sericoside, were isolated from the bark of Terminalia ivorensis. Their structures were established on the basis of 1D and 2D NMR data, chemical methods and tandem MS–MS spectrometry as a dimer of β-d-glucopyranosyl-18,19-seco-2α,3β,19,19,24-pentahydroxyolean-12-en-28-oate and β-d-glucopyranosyl-2α,3β,19α,24-tetrahydroxyolean-12-en-28-oate (ivorenoside A, 1), a dimer of β-d-glucopyranosyl-18,19-seco-24-carboxyl-2α,3β,19,19-tetrahydroxyolean-12-en-28-oate and β-d-glucopyranosyl-2α,3β,19α,24-tetrahydroxyolean-12-en-28-oate (ivorenoside B, 2) and β-d-glucopyranosyl-2α,3β,19β,24-tetrahydroxyolean-11-oxo-olean-12-en-28-oate (ivorenoside C, 3). Ivorenosides A and B are the first examples in nature of dimeric triterpenoid saponins with a 18,19-seco E ring of one of the two units. These isolated compounds were evaluated for their antioxidant properties and further for their cytotoxic activity against four human cancer cell lines. Ivorenoside B and C exhibited scavenging activity against DPPH and ABTS⁺ radicals with IC₅₀ values comparable with that of the standard drug Trolox and ivorenoside A showed antiproliferative activity against MDA-MB-231 and HCT116 human cancer cell lines with IC₅₀ values of 3.96 and 3.43 μM, respectively.     

The two beta forms of poly(L-glutamic acid).

β-Helical poly(L -glutamic acid) in a gel state was found to be easily converted to the antiparallel β form by heating. Two β forms were obtained, depending on the temperature of heating. Temperatures between 40° and 85°C produced a β form with a spacing between pleated sheets (d₀₀₁) of 9.03 Å, termed β₁. If the heating was carried out at temperatures higher than 85°C, the β₁ form underwent another conformational transition reducing the d₀₀₁ value from 9.03 to 7.83 Å (termed β₂) without any prominent change in the fiber repeat distance (i.e., the polypeptide backbone conformation). The time course of these two transitions was followed by measuring the infrared spectra of the samples, and it was concluded that the α → β₁ transition in its initial stage obeys a pseudo-first order rate process with activation enthalpy and entropy of 54 kcal/mol and 92 eu, respectively. On the other hand, the typical sigmoidal conversion curves observed for the transition between the two types of β forms (β₁ → β₂) indicate that this transition proceeds via a socalled “nucleation and growth” process. The kinetic theory of phase transitions developed by Avrami can be applied with success to explain this transition. The infrared spectra, in the region from 1800 to 200 cm^?1, were measured for these two β forms and the results showed that the conformation of the side chains and the mode of the hydrogen bonding between the side-chain carboxyl groups undergo appreciable change during the transition. The heat-induced conformational transition of poly(L -Glu⁷⁸ L -Val²²) was also studied. The copolymer was transformed from the α-helical conformation directly to the β₂ form. The reason for this was thought to be due to the fact that the L -valine residues and the L -glutamyl residues near the L -valine residues have a strong tendency to take the more compact β₂ form.     

A rapid assay for the binding of actinomycin to DNA.

The theory of countercurrent distribution (CCD) was reviewed and extended. The separation function for the fundamental distribution of CCD was presented in the form n = t²(αk₁+β)²/αk₁(β?1)² where n is the number of transfers, t the abscissa of the standard normal distribution, α = v_m/v₈ the phase ratio, β = k₁/k₂≥ 1 the separation factor, and k₁ the partition coefficient of the more radidly moving component; n was found to be minimal on the condition αk₁ = β. The separation function for the single withdrawal of CCD was obtained in the form N = u + 1 = t²{(αk₁ + 1)^1/2 + [β(αk₁ + β)]^1/2}²/(β ? 1)²+ 1, where N is the number of partition units. From this equation it appears that N is minimal when αk₁ = 0. Compared with the former separation functions presented in the literature, these separation functions have the advantage of giving directly the relationships among the phase ratio, the absolute partition coefficient, the separation factor, the resolution degree, and the number of transfers or partition units required. In addition, the dependencies of the elution volumes and the widths of the elution curves on α, β, and the partition coefficients were considered mathematically by means of differential calculus. The elution volumes were found to have minima at certain αk₁ values. The standard deviations, on the contrary, did not have minima in respect to αk₁. The theory presented can be used for selecting proper operating conditions while separating chemical compounds.     

Oxygen equilibria of hemoglobin A and hemoglobin S valency hybrids.

Oxygen equilibrium determinations with “unsymmetrical” MetHb/Hb hybrids derived from human hemoglobins A and S are reported. All four of the possible hybrids have higher oxygen affinity than the parent hemoglobins. The α₂^Metβ₂^S hybrid has a lower oxygen affinity than that of α₂^Metβ₂^S. However, both the β^Met hybrids have similar oxygen affinity. The Bohr value of α₂^Metβ₂^S is more negative than that of α₂^Metβ₂^A while the β^Met hybrids appear to have almost identical Bohr values. These findings favor the view that α and β chains in hemoglobin A have different conformations and indicate that hemoglobin S has a β-chain conformation different from that of β-chain of hemoglobin A. This difference is probably carried into the oxygenation properties of the α-chain in such a way as to be reflected only when the β chain is oxidized.     

Functional characterization of recombinant prefoldin complexes from a hyperthermophilic archaeon, Thermococcus sp. strain KS-1

Prefoldin is a heterohexameric molecular chaperone complex that is found in the eukaryotic cytosol and also in archaea. It captures a nonnative protein and subsequently delivers it to a group II chaperonin for proper folding. Archaeal prefoldin is a heterocomplex containing two α subunits and four β subunits with the structure of a double β-barrel assembly, with six long coiled coils protruding from it like a jellyfish with six tentacles. We have studied the protein folding mechanism of group II chaperonin using those of Thermococcus sp. strain KS-1 (T. KS-1) because they exhibit high protein folding activity in vitro. We have also demonstrated functional cooperation between T. KS-1 chaperonins and prefoldin from Pyrococcus horikoshii OT3. Recent genome analysis has shown that Thermococcus kodakaraensis KOD1 contains two pairs of prefoldin subunit genes, correlating with the existence of two different chaperonin subunits. In this study, we characterized four different recombinant prefoldin complexes composed of two pairs of prefoldin subunits (α1, α2, β1, and β2) from T. KS-1. All of them (α1-β1, α2-β1, α1-β2, and α2-β2) exist as α₂β₄ heterohexamers and can protect several proteins from forming aggregates with different activities. We have also compared the collaborative activity between the prefoldin complexes and the cognate chaperonins. Prefoldin complexes containing the β1 subunit interacted with the chaperonins more strongly than those with the β2 subunit. The results suggest that Thermococcus spp. express different prefoldins for different substrates or conditions as chaperonins.     

Partitioning taxonomic diversity of aquatic insect assemblages and functional feeding groups in neotropical savanna headwater streams

Biological diversity can be divided into: alpha (α, local), beta (β, difference in assemblage composition among locals), and gamma (γ, total diversity). We assessed the partitioning of taxonomic diversity of Ephemeroptera, Plecoptera and Trichoptera (EPT) and of functional feeding groups (FFG) in neotropical savanna (southeastern Brazilian cerrado) streams. To do so, we considered three diversity components: stream site (α), among stream sites (β₁), and among hydrologic units (β₂). We also evaluated the association of EPT genera composition with heterogeneity in land use, instream physical habitat structure, and instream water quality variables. The percentage of EPT taxonomic α diversity (20.7%) was smaller than the β₁ and β₂ diversity percentages (53.1% and 26.2%, respectively). The percentage of EPT FFG collector-gatherer α diversity (26.5%) was smaller than that of β₁ diversity (55.8%) and higher than the β₂ (17.7%) diversity. The collector-gatherer FFG was predominant and had the greatest β diversity percentage among stream sites (β₁, 55.8%). Our findings support the need for implementing regional scale conservation strategies in the cerrado biome, which has been degraded by anthropogenic activities.     

Comparison of the activation energy barrier for succinimide formation from α- and β-aspartic acid residues obtained from density functional theory calculations

The l-α-Asp residues in peptides or proteins are prone to undergo nonenzymatic reactions to form l-β-Asp, d-α-Asp, and d-β-Asp residues via a succinimide five-membered ring intermediate. From these three types of isomerized aspartic acid residues, particularly d-β-Asp has been widely detected in aging tissue. In this study, we computationally investigated the cyclization of α- and β-Asp residues to form succinimide with dihydrogen phosphate ion as a catalyst (H₂PO₄⁻). We performed the study using B3LYP/6-31 + G(d,p) density functional theory calculations. The comparison of the activation barriers of both residues is discussed. All the calculations were performed using model compounds in which an α/β-Asp-Gly sequence is capped with acetyl and methylamino groups on the N- and C-termini, respectively. Moreover, H₂PO₄⁻ catalyzes all the steps of the succinimide formation (cyclization-dehydration) acting as a proton-relay mediator. The calculated activation energy barriers for succinimide formation of α- and β-Asp residues are 26.9 and 26.0 kcal mol^− 1, respectively. Although it was experimentally confirmed that β-Asp has higher stability than α-Asp, there was no clear difference between the activation barriers. Therefore, the higher stability of β-Asp residue than α-Asp residue may be caused by an entropic effect associated with the succinimide formation.     

Specific loops D,E and F of nicotinic acetylcholine receptor β1 subunit may confer imidacloprid selectivity between Myzus persicae and its predatory enemy Pardosa pseudoannulata

One nicotinic acetylcholine receptor non-α subunit was cloned from the pond wolf spider, Pardosa pseudoannulata, an important predatory enemy of some insect pests with agricultural importance, such as the green peach aphid Myzus persicae. The subunit shows high amino acid identities to insect β1 subunits (74–78%), and was denoted as Ppβ1. Although high identities are found between Ppβ1 and insect β1 subunits, amino acid differences are found within loops D, E and F, important segments contributing to ligand binding. The effects of amino acid differences within these loops were evaluated by introducing loops of insect or spider β1 subunits into rat β2 subunit and co-expressing with insect α subunit. The corresponding regions of rat β2 chimera β2^Mpβ1 (β2 with loops D, E and F from M. persicae β1 subunit Mpβ1) were replaced by loops D, E and F of Ppβ1 singly or together to construct different chimeras. When these chimeras were co-expressed with insect Nlα1, it was found that the replacement of loops D, E and F of β2^Mpβ1 by that of Ppβ1 resulted in a right-ward shift of the imidacloprid dose–response curves, reflecting increases in EC₅₀, compared to Nlα1/β2^Mpβ1. By contrast, the influences on ACh potency were minimal. The further study showed that R81Q, N137G and F190W differences, within loops D, E and F respectively, contributed mainly to these sensitivity changes. This study contributes to our understanding of the molecular mechanism underlying selectivity of neonicotinoids against insects over spiders.     

Relationship between β4 hydrogen bond and β6 hydrophobic interactions during aggregate, fiber or crystal formation in oversaturated solutions of hemoglobin A and S

Oversaturated deoxy-α₂β₂^T4V aggregated instantly without a delay time, which is in contrast to the delay time before the generation of fibers of deoxy-HbS and deoxy-α₂β₂^E6V,D73H. Solubility of deoxy-α₂β₂^T4V was ∼10-fold lower than that of deoxy-HbS and was similar to oxy- and deoxy-α₂β₂^E6V,T4V. These results indicate that β4Val in HbA in the oxy and deoxy forms with or without β6Val facilitates hydrophobic interaction of the A-helix with the EF helix of adjacent molecules without forming a β4/β73 hydrogen bond. Deoxy-HbA generated crystals following aggregation as does HbC-Harlem(α₂β₂^E6V,D73N), while α₂β₂^T4V and α₂β₂^D73H as well as HbS, α₂β₂^E6V,D73H and α₂β₂^E6V,T4V in the oxy and deoxy forms did not form crystals, indicating in addition to the strength of β6 amino acid hydrophobicity that the synergism between the β4Thr hydrogen bond and β6 hydrophobic interaction free energies on the A-helix play a critical role in formation of fibers versus crystalline nuclei during phase transition.     

Soret Spectroscopic and Molecular Graphic Analysis of Human Semi-β-Hemoglobin Formation

The interaction of heme-free α (α^o) and heme-containing β (β^h) chains of human hemoglobin has been monitored in 0.1 M potassium phosphate buffer, pH 7 or 8, at [5°C. Soret zero and first-derivative spectra were consistent with a uniform association reaction. Stopped-flow investigations demonstrated association rates on the order of 10⁷ M^?1 s^?1. This was 100-fold more rapid than the reported rate of combination of α^h and β^h proteins. This encounter-like rate of semi-β-hemoglobin (α^oβ^h) formation was increased by raising the pH from 7 to 8. pH change is known to affect the spatial arrangement of AB—GH helical entities. Molecular graphic analysis of modeled α^o protein superimposed over native α^h protein revealed an apo Mb-like structure with well-defined AB—GH segments. Repositioning of these core helical segments, resulting in increased conformational freedom of the α₁β₁ interface, was apparently responsible for the enhanced association properties of the α^o protein.     

Broadband dielectric spectroscopy and calorimetric investigations of d-lyxose

Using broadband dielectric spectroscopy, we have studied different types of relaxation processes, namely, primary (α), secondary (β), and another sub-T_g process called γ-process, in the supercooled state of d-lyxose, over a wide frequency (10^-2–) and temperature range (120–340 K). In addition, the same sample was analyzed by differential scanning calorimeter. The temperature dependence of the relaxation times as well as the dielectric strength of different processes has been critically examined. It has been observed that the slower secondary relaxation (designated as β-) process shifts to lower frequencies with increasing applied pressure, but not the faster one. This pressure dependence indicates that the observed slower secondary relaxation (β-) is Johari–Goldstein relaxation process and faster one (γ-process) is probably the rotation of hydroxymethyl (–CH₂OH) side group attached to the sugar ring, that is, of intramolecular origin.     

Dependence of the energy lifetime on the type of anomalous losses in stellarators

A relatively simple model of transport process in stellarators that was proposed earlier by the author on the basis of neoclassical theory makes it possible to determine the density and temperature profiles of the plasma components, the ambipolar electric field profile, and the particle and energy lifetimes from the given device parameters and given particle and energy sources with allowance for anomalous losses. The results of numerical simulations carried out with this model for the L-2M, ATF, CHS, and LHD stellarators over broad ranges of plasma densities and absorbed powers showed that the plasma energy lifetimes in these devices coincide to within factors on the order of two with those found from empirical scalings. A specific model of anomalous losses was chosen for calculations. Results are presented from simulations with a more general form of the anomalous thermal conductivity. Namely, the thermal conductivity is chosen to be K _j ^(a) ≈ N ^α T _j ^β B ₀ ^?γ , where N(r) is the plasma density and T _j (r) is the temperature of the jth plasma component (j = e, i). The parameters α, β, and γ are set equal to α = 1, β = 2, and γ = 1; α = 0.5, β = 2.5, and γ = 1; α = 1.5, β = 2, and γ = 2; α = 1, β = 2.5, and γ = 2; and α = 1.5, β = 2.5, and γ = 2. The simulations have been done for the L-2M and LHD stellarators. It is found that, in all the five models, the calculated energy lifetimes τ _c are essentially independent of the functional form of the anomalous thermal conductivity and coincide to within a factor on the order of two with those following from the LHD scaling.     

Roles for the two N-terminal (β/α) modules in the folding of a (β/α)₈-barrel protein as studied by fragmentation analysis

The (β/α)₈‐barrel is one of the most abundant folds found in enzymes. To identify the independent folding units and the segment(s) that correspond to a minimum core structure within a (β/α)₈‐barrel protein, fragmentation experiments were performed with Escherichia coli phosphoribosylanthranilate isomerase, which has a single (β/α)₈‐barrel domain. Our previous studies indicated that the central four β/α segments comprise an independent folding unit; whereas, the role(s) of the first two β/α segments in folding had not been clarified prior to this report. Herein, we report the design and synthesis of a series of N‐terminally deleted fragments starting with (β/α)_1–5β₆ as the parent construct. Analytical gel filtration and urea‐induced equilibrium unfolding experiments indicated that deletions within the N‐terminal region, that is, within the first two β/α modules, resulted in reduced stability or aggregation of the remaining segments. The (β/α)_3–5β₆ segment appeared to fold into a stable structure and deletion of β₆ from (β/α)_3–5β₆ yielded (β/α)_3–5, which did not form native‐like secondary structures. However, urea‐induced unfolding of (β/α)_3–5, monitored by reduction of tryptophan fluorescence, indicated that the fragment contained a loosely packed hydrophobic core. Taken together, the results of our previous and present fragmentation experiments suggest the importance of the central (β/α)_3–4β₅ module in folding, which is a finding that is compatible with our simulated unfolding study performed previously. Proteins 2010. © 2010 Wiley‐Liss, Inc.