What should the Z-score of native protein structures be?

The Z-score of a protein is defined as the energy separation between the native fold and the average of an ensemble of misfolds in the units of the standard deviation of the ensemble. The Z-score is often used as a way of testing the knowledge-based potentials for their ability to recognize the native fold from other alternatives. However, it is not known what range of values the Z-scores should have if one had a correct potential. Here, we offer an estimate of Z-scores extracted from calorimetric measurements of proteins. The energies obtained from these experimental data are compared with those from computer simulations of a lattice model protein. It is suggested that the Z-scores calculated from different knowledge-based potentials are generally too small in comparison with the experimental values.     

The γ subunits of the native GABAA/benzodiazepine receptors

Subunit-specific antibodies to all the γ subunit isoforms described in mammalian brain (γ₁, γ_2S, γ_L, and γ₃) have been made. The proportion of GABA_A receptors containing each γ subunit isoform in various brain regions has been determined by quantitative immunoprecipitation. In all tested regions of the rat brain, the γ₁, and γ₃ subunits are present in considerable smaller proportion of GABA_A receptor than the γ₂ subunit. Immunocytochemistry shows that γ₁ immunoreactivity concentrates in the stratum oriens and stratum radiatum of the CA1 region of the hippocampus. In the dentate gyrus, γ₁ immunoreactivity concentrates on the outer 2/3 of the molecular layer coinciding with the localization of the axospinous synapses of the perforant pathway. In contrast, γ₃ immunoreactivity concentrates on the basket cells and other GABAergic local circuit neurons of the hilus. These cells are also rich in γ_2S. In the cerebellu, γ₁ immunolabeling was localized on the Bergmann glia. The γ_2S and γ_2L subunits are differentially expressed in various brain regions. Thus the γ_2S is highly expressed in the olfactory bulb and hippocampus whereas the γ_2L is very abundant in inferior colliculus and cerebellum, particularly in Purkinje cells, as immunocytochemistry, in situ hybridization and immunoprecipitation techniques have revealed. The γ_2S and γ_2L coexist in some brain areas and cell types. Moreover, the γ_2S and γ_2L subunits can coexist in the same GABA_A receptor pentamer. We have shown that this is the case in some GABA_A receptors expressed in cerebellar granule cells. These GABA_A receptors also have α and β subunits forming the pentamer. Immunoblots have shown that the rat γ₁, γ_2S, γ_2L and γ₃ subunits are peptides of 47, 45, 47 and 44 kDa respectively. Results also indicate that there are aging-related changes in the expression of the γ_2S and γ_2L subunits in various brain regions which suggest the existence of aging-related changes in the subunit composition of the GABA_A receptors which in turn might lead to changes in receptor pharmacology. The results obtained with the various γ subunit isoforms are discussed in terms of the high molecular and binding heterogeneity of the native GABA_A receptors in brain. Special issue dedicated to Dr. Kinya Kuriyama     

Habitat enhancement and native fish conservation: can enhancement of channel complexity promote the coexistence of native and introduced fishes?

Native fishes worldwide have declined as a consequence of habitat loss and degradation and introduction of non-native species. In response to these declines, river restoration projects have been initiated to enhance habitat and remove introduced fishes; however, non-native fish removal is not always logistically feasible or socially acceptable. Consequently, managers often seek to enhance degraded habitat in such a way that native fishes can coexist with introduced species. We quantified dynamics of fish communities to three newly constructed side channels in the Provo River, Utah, USA, to determine if and how they promoted coexistence between native fishes (nine species) and non-native brown trout (Salmo trutta L.). Native and introduced fishes responded differently in each side channel as a function of the unique characteristics and histories of side channels. Beaver activity in two of the three side channels caused habitat differentiation or channel isolation that facilitated the establishment of native species. The third side channel had greater connectivity to and similar habitat as the main channel of the Provo River, resulting in a similar fish community to main channel habitats (i.e. dominated by brown trout with only a few native fish species). These results demonstrate the importance of understanding habitat preferences for each species in a community to guide habitat enhancement projects and the need to create refuge habitats for native fishes.     

Aspects of the mineral nutrition of some native British plants — inter-site variation

Fifteen common native British plants were each sampled at a range of sites in Great Britain and green tissues analysed for several inorganic nutrients. Sampling criteria are discussed. The inter-site variation of each element within a species is assessed as a frequency distribution of raw data. Sample values of parameters including arithmetic mean, variance (coefficient of variation), skewness and kurtosis are presented. Their stability is assessed from nitrogen in sub-samples of Pteridium. This suggested sample sizes were adequate but some distributions had sufficient kurtosis to affect the variance. These parameters showed distinctions between macro- and micro-elements and between species. Some mean values sharply distinguished between species and may help to assess current theories of strategy and adaptation but a wider range of species is needed to clarify trends. Coefficients of variation are discussed and were relatively low for a nation-wide survey after allowing for sampling constraints. Coefficients of skewness and kurtosis showed two-thirds of the sample distributions were non-normal. Ecological aspects of the distributions are discussed and are relevant to studies along environmental gradients. Published hypotheses linking positive skewness to stress in the field are considered and two other postulates discussed. Distribution bounds such as those confining 95% of the values are discussed in relation to possible critical levels of nutrients.Nomenclature follows Clapham et al. (1981), Excursion flora of the British Isles. 3rd ed. University Press, Cambridge, except Chamaenerion.     

How does sucrose stabilize the native state of globular proteins?

It is well known that sucrose stabilizes the native state of globular proteins against both chemical denaturants and temperature. A largely accepted explanation of sucrose-induced stabilization is not yet emerged. It is shown that the same theoretical approach able to rationalize the occurrence of cold denaturation, the contrasting role of GdmCl and Gdm₂SO₄, and the TMAO counteraction of urea denaturing activity [PCCP 12 (2010) 14245; PCCP 13 (2011) 12008; PCCP 13 (2011) 17689] works well also in the case of sucrose. The solvent-excluded volume effect plays the fundamental role because sucrose addition to water causes a marked increase in volume packing density due to the large size of sucrose molecules, that act as crowding agents.     

Biochemical characterization of the native α-carbonic anhydrase purified from the mantle of the Mediterranean mussel,Mytilus galloprovincialis

A α-carbonic anhydrase (CA, EC 4.2.1.1) has been purified and characterized biochemically from the mollusk Mytilus galloprovincialis. As in most mollusks, this α-CA is involved in the biomineralization processes leading to the precipitation of calcium carbonate in the mussel shell. The new enzyme had a molecular weight of 50?kDa, which is roughly two times higher than that of a monomeric α-class enzyme. Thus, Mytilus galloprovincialis α-CA is either a dimer, or similar to the Tridacna gigas CA described earlier, may have two different CA domains in its polypeptide chain. The Mytilus galloprovincialis α-CA sequence contained the three His residues acting as zinc ligands and the gate-keeper residues present in all α-CAs (Glu106-Thr199), but had a Lys in position 64 and not a His as proton shuttling residue, being thus similar to the human isoform hCA III. This probably explains the relatively low catalytic activity of Mytilus galloprovincialis α-CA, with the following kinetic parameters for the CO₂ hydration reaction: k_cat =?4.1?×?10⁵ s^?1 and k_cat/K_m of 3.6?×?10⁷ M^?1 × s^?1. The enzyme activity was poorly inhibited by the sulfonamide acetazolamide, with a K_I of 380?nM. This study is one of the few describing in detail the biochemical characterization of a molluskan CA and may be useful for understanding in detail the phylogeny of these enzymes, their role in biocalcification processes and their potential use in the biomimetic capture of the CO₂.     

Spectroscopic characterization of the inclusion complexes of luteolin with native and derivatized β-cyclodextrin

The inclusion complexes of Luteolin (LU) with cyclodextrins (CDs) including β-cyclodextrin (βCD), hydroxypropyl-β-cyclodextrin (HPβCD) and dimethyl-β-cyclodextrin (DMβCD), Scheme 1, have been investigated using the method of steady-state fluorescence. The stoichiometric ratio of the three complexes was found to be 1:1 and the stability constants (K) were estimated from spectrofluorometric titrations, as well as the thermodynamic parameters. Maximum inclusion ability was obtained in the case of HPβCD followed by DMβCD and βCD. Moreover, ¹H NMR and 2D NMR were carried out, revealing that LU has different form of inclusion which is in agreement with molecular modeling studies. These models confirm that when LU–βCD and LU–DMβCD complexes are formed, the B-ring is oriented toward the primary rim; however, for LU–HPβCD complex this ring is oriented toward the secondary rim. The ESR results showed that the antioxidant activity of luteolin was the order LU–HPβCD > LU–DMβCD > LU–βCD > LU, hence the LU-complexes behave are better antioxidants than luteolin free.     

Is the isolated ligand binding domain a good model of the domain in the native receptor?

Numerous studies have used the atomic level structure of the isolated ligand binding domain of the glutamate receptor to elucidate the agonist-induced activation and desensitization processes in this group of proteins. However, no study has demonstrated the structural equivalence of the isolated ligand binding fragments and the protein in the native receptor. In this report, using visible absorption spectroscopy we show that the electronic environment of the antagonist 6-cyano-7-nitro-2,3-dihydroxyquinoxaline is identical for the isolated protein and the native glutamate receptors expressed in cells. Our results hence establish that the local structure of the ligand binding site is the same in the two proteins and validate the detailed structure-function relationships that have been developed based on a comparison of the structure of the isolated ligand binding domain and electrophysiological consequences in the native receptor.     

An investigation of the action of porcine pancreatic α-amylase on native and gelatinised starches

The action of pancreatic α-amylase (EC 3.2.1.1) on various starches has been studied in order to achieve better understanding of how starch structural properties influence enzyme kinetic parameters. Such studies are important in seeking explanations for the wide differences reported in postprandial glycaemic and insulinaemic indices associated with different starchy foodstuffs. Using starches from a number of different sources, in both native and gelatinised forms, as substrates for porcine α-amylase, we showed by enzyme kinetic studies that adsorption of amylase to starch is of kinetic importance in the reaction mechanism, so that the relationship between reaction velocity and enzyme concentration [E₀] is logarithmic and described by the Freundlich equation. Estimations of catalytic efficiencies were derived from measurements of k_cat/K_m performed with constant enzyme concentration so that comparisons between different starches were not complicated by the logarithmic relationship between E₀ and reaction velocity. Such studies reveal that native starches from normal and waxy rice are slightly better substrates than those from wheat and potato. After gelatinisation at 100°C, k_cat/K_m values increased by 13-fold (waxy rice) to 239-fold (potato). Phosphate present in potato starch may aid the swelling process during heating of suspensions; this seems to produce a very favourable substrate for the enzyme. Investigation of pre-heat treatment effects on wheat starch shows that the relationship between treatment and k_cat/K_m is not a simple one. The value of k_cat/K_m rises to reach a maximum at a pre-treatment temperature of 75°C and then falls sharply if the treatment is conducted at higher temperatures. It is known that amylose is leached from starch granules during heating and dissolves. On cooling, the dissolved starch is likely to retrograde and become resistant to amylolysis. Thus the catalytic efficiency tends to fall. In addition, we find that the catalytic efficiency on the different starches varies inversely with their solubility and we interpret this finding on the assumption that the greater the solubility, the greater is the likelihood of retrogradation. We conclude that although α-amylase is present in high activity in digestive fluid, the enzymic hydrolysis of starch may be a limiting factor in carbohydrate digestion because of factors related to the physico-chemical properties of starchy foods.     

Does the recruitment of a non‐native mussel in native eelgrass habitat explain their disjunct adult distributions?

We studied variability in the abundance of small individuals of an invasive mussel (Musculista senhousia) across the depth distribution of a native marine angiosperm, eelgrass (Zostera marina). Adult mussels and eelgrass have a disjunct local distribution, each limiting the other in complex ways. To assess whether eelgrass also influenced the distribution of juvenile mussels, we sampled inside and outside eelgrass beds in one site in Mission Bay and two in San Diego Bay, California, USA. We sampled mussels in size classes 0.26–0.50 mm, 0.51–1.00 mm, 1.10–2.00 mm and > 2.00 mm from September 1997 to April 1999. We also monitored gonad development in larger mussels and in situ growth of mussels ≤ 2 mm tagged with the chemical marker calcein. Spatial and temporal variations in mussel abundances were high but seasonal patterns were roughly similar at San Diego Bay sites; very few mussels were found in Mission Bay. Mussels with full gonads were found year‐round in San Diego Bay, as were mussels in the smallest size class (with a large peak in fall and a smaller secondary one in spring), suggesting that many of the smallest mussels represent recruitment. The observation that most, although not all, tagged mussels increased in size provides further support for recruitment. Some of the highest numbers of mussels in the smallest size class were found inside eelgrass beds, indicating that eelgrass does not restrict and may actually enhance the distribution of very small mussels. The disjunct distribution of adult mussels and eelgrass thus is apparently established primarily postrecruitment. M. senhousia is capable of year‐round reproduction, recruitment and growth, and thus is poised to preempt space from eelgrass following any disturbance that results in eelgrass declines, such as habitat fragmentation, eutrophication, or disease.     

Can the introduction of Xenopus laevis affect native amphibian populations? Reduction of reproductive occurrence in presence of the invasive species

Biological invasions are regarded as a form of global change and potential cause of biodiversity loss. Xenopus laevis is an anuran amphibian native to sub-Saharan Africa with strong invasive capacity, especially in geographic regions with a Mediterranean climate. In spite of the worldwide diffusion of X. laevis, the effective impact on local ecosystems and native amphibian populations is poorly quantified. A large population of X. laevis occurs in Sicily and our main aim of this work was to assess the consequences of introduction of this alien species on local amphibian populations. In this study we compare the occurrence of reproduction of native amphibians in ponds with and without X. laevis, and before and after the alien colonization. The results of our study shows that, when X. laevis establishes a conspicuous population in a pond system, the populations of Discoglossus pictus, Hyla intermedia and Pelophylax synklepton esculentus show clear signs of distress and the occurrence of reproduction of these native amphibians collapses. In contrast, the populations of Bufo bufo do not appear to be affected by the alien species. Since the Sicilian population of X. laevis shows a strong dispersal capacity, proportionate and quick interventions become necessary to bound the detriment to the Sicilian amphibians populations.     

Influence of organization of native protein structure on its folding: Modeling of the folding of α-helical proteins

An important question that is addressed here is whether the modeling of protein folding can catch the difference between the folding of proteins with similar structures but with different folding mechanisms. In this work, the modeling of folding of four α-helical proteins from the homeodomain family, which are similar in size, was done using the Monte Carlo and dynamic programming methods. A frequently observed order of folding of α-helices for each protein was determined using the Monte Carlo method. A correlation between the experimental folding rate and the number of Monte Carlo steps was also demonstrated. Amino acid residues that are important for the folding were determined using the dynamic programming method. The defined regions correlate with the order of folding of secondary-structure elements in the proteins both in experiments and in modeling.     

Diverse effects on the native β-sheet of the human prion protein due to disease-associated mutations

Prion diseases are fatal neurodegenerative disorders that involve the conversion of the normal cellular form of the prion protein (PrP(C)) to a misfolded pathogenic form (PrP(Sc)). There are many genetic mutations of PrP associated with human prion diseases. Three of these point mutations are located at the first strand of the native β-sheet in human PrP: G131V, S132I, and A133V. To understand the underlying structural and dynamic effects of these disease-causing mutations on the human PrP, we performed molecular dynamics of wild-type and mutated human PrP. The results indicate that the mutations induced different effects but they were all related to misfolding of the native β-sheet: G131V caused the elongation of the native β-sheet, A133V disrupted the native β-sheet, and S132I converted the native β-sheet to an α-sheet. The observed changes were due to the reorientation of side chain-side chain interactions upon introducing the mutations. In addition, all mutations impaired a structurally conserved water site at the native β-sheet. Our work suggests various misfolding pathways for human PrP in response to mutation.     

Chaperone-mediated native folding of a β-scorpion toxin in the periplasm of Escherichia coli

Background

Animal neurotoxin peptides are valuable probes for investigating ion channel structure/function relationships and represent lead compounds for novel therapeutics and insecticides. However, misfolding and aggregation are common outcomes when toxins containing multiple disulfides are expressed in bacteria.

Methods

The β-scorpion peptide toxin Bj-xtrIT from Hottentotta judaica and four chaperone enzymes (DsbA, DsbC, SurA and FkpA) were co-secreted into the oxidizing environment of the Escherichia coli periplasm. Expressed Bj-xtrIT was purified and analyzed by HPLC and FPLC chromatography. Its thermostability was assessed using synchrotron radiation circular dichroism spectroscopy and its crystal structure was determined.

Results

Western blot analysis showed that robust expression was only achieved when cells co-expressed the chaperones. The purified samples were homogenous and monodisperse and the protein was thermostable. The crystal structure of the recombinant toxin confirmed that it adopts the native disulfide connectivity and fold.

Conclusions

The chaperones enabled correct folding of the four-disulfide-bridged Bj-xtrIT toxin. There was no apparent sub-population of misfolded Bj-xtrIT, which attests to the effectiveness of this expression method.

General significance

We report the first example of a disulfide-linked scorpion toxin natively folded during bacterial expression. This method eliminates downstream processing steps such as oxidative refolding or cleavage of a fusion-carrier and therefore enables efficient production of insecticidal Bj-xtrIT. Periplasmic chaperone activity may produce native folding of other extensively disulfide-reticulated proteins including animal neurotoxins. This work is therefore relevant to venomics and studies of a wide range of channels and receptors.     

Do trophic subsidies affect the outcome of introductions of a non‐native freshwater fish?

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Phylogenetic analysis of the swine leukocyte antigen — 2 gene for Korean native pigs

The objective of this study was to investigate genetic relationships of the SLA-2 gene, to characterize SLA-2 alleles, and to provide basic genetic information of Korean pigs. The swine leukocyte antigen - 2 (SLA-2) gene in the MHC classical region was cloned with spleen tissues from Korean native pigs selected from the main land (KNP) and Jeju Island (KJP). Primer sequences based on swine cDNA (GenBank accession numbers AF464049 and AF464005) were used to amplify the entire SLA-2 gene, and the amplification product including both 3′ and 5′ UTRs was sized 1,520 bp. A BAC clone was selected from miniature pigs and sequenced for the genomic region of the SLA-2 gene showing that 4,585 bp in total length consisted of exons (1,087 bp) and introns (3,498 bp). A sequence analysis confirmed 58 SNPs in coding regions, which revealed higher numbers of SNPs in KNP than other pig breeds, implicating more genetic variability in Korean pigs. Approximately 82% of the SLA-2 SNPs were located in the highly polymorphic exons 2 and 3. Newly identified sequences of the SLA-2 gene for KNP and KJP were submitted into the IPD-MHC database with new nomenclatures (SLA-2*1501, SLA-2*1601, and SLA-2*w08hy01 allele), while the representative sequences of KNP and KJP were submitted into GenBank with accession numbers (DQ992495, DQ992496, and DQ992501), respectively. The identified KNP allele (SLA-2*1501) clustered with previously defined alleles for Korean pigs (SLA-2*kn02 and SLA-2*jh01), but SLA-2*1601 (KNP) and SLA-2*w08hy01 (KJP) alleles showed no significant genetic relationships with any other allele. A sequence comparison revealed that KNP has departed from KJP both genetically and phenotypically. The results of SLA-2 SNP in KNP and KJP reported here will serve as the SLA-2 reference for Korean pigs.