The structure of 5a,6–anhydrotetracycline and its Mg2+ complexes in aqueous solution

Semiempirical molecular orbital theory has been used for a systematic scan of the binding positions for a Mg²⁺ ion with 5a,6–anhydrotetracycline taking both conformational flexibility and possible different tautomeric forms into account. The magnesium ion has been calculated alone and with four or five complexed water molecules in order to simulate the experimental situation more closely. The results are analyzed by comparing the behavior of the title compound with that of tetracycline itself and possible causes for the stronger induction of the Tetracycline Receptor (TetR) by 5a,6–anhydrotetracycline than by tetracycline are considered. Energetically favored 3D -structure of the zwitteranionic 5a,6-anhydrotetracycline magnesium complex in solution Electronic Supplementary Material Supplementary material is available for this article at     

Pyruvate kinase M2 in chronic inflammations: a potpourri of crucial protein–protein interactions

Cell Biology and Toxicology - Chronic inflammation (CI) is a primary contributing factor involved in multiple diseases like cancer, stroke, diabetes, Alzheimer’s disease, allergy, asthma,...     

Angular disorder in actin: is it consistent with general principles of protein structure?

Harold Erickson has recently provided a useful analysis of helical structures having one class versus two classes of intersubunit bonds. His analysis is based upon an assumption that the subunits themselves are essentially unchanged upon bond formation (polymerization). He shows that such a structure having two classes of bonds (i.e. one in which each subunit interacts with four of its neighbors rather than two) can explain some of the features of actin. While he acknowledges that for actin there could be a conformational change and that, in principle, it could explain such features, he argues that the allowed magnitude of such a conformational change is inadequate. Since kinetics and thermodynamics cannot distinguish between the energy derived from the formation of a bond from that due to a conformational change, the question of whether the features of F-actin are derived from a conformational change or a system of two classes of bonds or both must be answered with high-resolution structural information. Recent studies by K. C. Holmes and others suggest that the second possibility might be closest to the truth. The heart of our disagreement is not whether Erickson's thermodynamic analysis is correct, given rigid subunits, but whether all protein polymers are characterized by rigid subunits with rigid intersubunit contacts. Erickson maintains that the observation of an angular disorder of 12 degrees per subunit within the actin filament conflicts with his formalism of rigid subunit interfaces and must therefore result from the erroneous interpretation of measurements. He presents an alternative model to explain the observations. His model, however, does not account for the observations and we will argue that, ultimately, like the resolution of the matter of the number of classes of bonds and the extent of their contact, the amount of angular disorder will require higher-resolution structural studies.     

Split target specificity of ResT: a design for protein delivery,site selectivity and regulation of enzyme activity?

The ResT telomere resolvase is responsible for maintaining the hairpin telomeres that cap the linear chromosome and minichromosomes of Borrelia burgdorferi. This enzyme acts at the tandem telomere junctions present within circular dimers resulting from DNA replication. ResT mediates the transesterification steps of resolution using a constellation of active site residues similar to that found in tyrosine recombinases and type IB topoisomerases. By combining this reaction mechanism with a hairpin binding module in its N-terminal domain, ResT reduces a fused telomere dimer into two hairpin monomers. ResT displays a split DNA binding specificity, with the N- and C-terminal domains targeting distinct regions of the telomere. This bi-specificity in binding is likely to be important in protein delivery, substrate selection and regulation of enzyme activity.     

Synthesis and disulfide bond connectivity–activity studies of a kalata B1-inspired cyclopeptide against dengue NS2B–NS3 protease

Kalata B1 is a plant protein with remarkable thermal, chemical and enzymatic stability. Its potential applications could be centered on the possibility of using its cyclic structure and cystine knot motif as a scaffold for the design of stable pharmaceuticals. To discover potent dengue NS2B–NS3 protease inhibitors, we have prepared various kalata B1 analogues by varying the amino acid sequence. Mass spectrometric and biochemical investigations of these analogues revealed a cyclopeptide whose two fully oxidized forms are substrate-competitive inhibitors of the dengue viral NS2B–NS3 protease. Both oxidized forms showed potent inhibition with K_i of 1.39 ± 0.35 and 3.03 ± 0.75 μM, respectively.     

The structure, properties, and nature of unconventional π halogen bond in the complexes of Al 4 2- and halohydrocarbons

Quantum chemical calculations have been performed to study the all-metal π halogen bonding in Al(4)(2-)···halohydrocarbon complexes. The result shows the existence of the all-metal π halogen bond in the complexes. There are three interaction modes (top, corner, and side) between Al(4)(2-) and halohydrocarbon. The interaction energy of this interaction varies from a positive value to -90.54 kJ mol(-1) in Al(4)(2-)···I-ethyne-s complex. The interaction strength is affected greatly by the hybridization of C atom and follows the order of C(sp(3)) < C(sp(2)) < C(sp) in most complexes. The methyl group in the halogen donor plays a negative contribution to the formation of halogen bond. The halogen bonding becomes stronger for the heavier halogen atom. The effect of binding site on the strength of halogen bond is related with the nature of halogen atom. The complexes have been analyzed with electrostatic potential, NICS, ELF, NBO, and AIM.     

Curative and protective activity in rabbits after reinfection with Schistosoma mansoni: a new model of immunity?

New Zealand rabbits were infected on day 1 and challenged on days 15, 30, and 60 with 1,000 Schistosoma mansoni cercariae/animal/infection. Challenged and control rabbits were perfused 60 days after each infection, corresponding to days 75, 90, and 120 after the first exposure. No decrease in number of adult schistosomes occurred in animals reinfected 15 days after primary infection, but, when the rabbits were challenged 30 and 60 days after the first infection, worm burden reduction of 61.4% and 92.6%, respectively, was observed as compared to infection controls. These data indicate that rabbits submitted to reinfection are able to kill the worms from their primary infection, besides being protected against challenge parasites.     

Pancreatic beta-cell growth and survival in the onset of type 2 diabetes: a role for protein kinase B in the Akt?

The control of pancreatic beta-cell growth and survival in the adult plays a pivotal role in the pathogenesis of type 2 diabetes. In certain insulin-resistant states, such as obesity, the increased insulin-secretory demand can often be compensated for by an increase in beta-cell mass, so that the onset of type 2 diabetes is avoided. This is why approximately two-thirds of obese individuals do not progress to type 2 diabetes. However, the remaining one-third of obese subjects that do acquire type 2 diabetes do so because they have inadequate compensatory beta-cell mass and function. As such, type 2 diabetes is a disease of insulin insufficiency. Indeed, it is now realized that, in the vast majority of type 2 diabetes cases, there is a decreased beta-cell mass caused by a marked increase in beta-cell apoptosis that outweighs rates of beta-cell mitogenesis and neogenesis. Thus a means of promoting beta-cell survival has potential therapeutic implications for treating type 2 diabetes. However, understanding the control of beta-cell growth and survival at the molecular level is a relatively new subject area of research and still in its infancy. Notwithstanding, recent advances have implicated signal transduction via insulin receptor substrate-2 (IRS-2) and downstream via protein kinase B (PKB, also known as Akt) as critical to the control of beta-cell survival. In this review, we highlight the mechanism of IRS-2, PKB, and anti-apoptotic PKB substrate control of beta-cell growth and survival, and we discuss whether these may be targeted therapeutically to delay the onset of type 2 diabetes.     

Elucidation of binding mode and three dimensional quantitative structure–activity relationship studies of a novel series of protein kinase B/Akt inhibitors

Protein kinase B (PKB; also known as Akt kinase) is located downstream in the PI-3 kinase pathway. Overexpression and constitutive activation of PKB/Akt leads to human prostate, breast and ovarian carcinomas. A series of 69 PKB/Akt inhibitors were examined to explore their binding modes using FlexX, and three-dimensional quantitative structure–activity relationship (3D-QSAR) studies based on comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were performed to provide structural insights into these compounds. CoMFA produced statistically significant results, with cross-validated q ² and non-cross validated correlation r ² coefficients of 0.53 and 0.95, respectively. For CoMSIA, steric, hydrophobic and hydrogen bond acceptor fields jointly yielded ‘leave one out’ q ²  = 0.51 and r ²  = 0.84. The predictive power of CoMFA and CoMSIA was determined using a test set of 13 molecules, which gave correlation coefficients, of 0.58 and 0.62, respectively. Molecular docking revealed that the binding modes of these molecules in the ATP binding sites of the Akt kinase domain were very similar to those of the co-crystallized ligand. The information obtained from 3D contour maps will allow the design of more potent and selective Akt kinase inhibitors.     

Isolation,structure determination and biological activity of A-16686 factors A′ 1, A′ 2 and A′ 3 glycolipodepsipeptide antibiotics

Summary WhenActinoplanes strain ATCC 33076, the producer of A-16686 A1, A2 and A3 complex, is fermented in a suitable medium three additional factors, designated A1, A2 and A3 are produced. These were isolated and characterized, and were shown to differ from the parent components of the original complex by lacking one mannose unit. Bioconversion of A factors into A factors was achieved by incubation with the mycelium ofActinoplanes ATCC 33076. Factor A2 has better antibacterial activity than A2 against some bacteria.     

The importance of protein–protein interactions for optimising oxygen activity in photosystem II: reconstitution with a recombinant thioredoxin—manganese stabilising protein

In this paper we describe how photosystem II (PSII) from higher plants, which have been depleted, of the extrinsic proteins can be reconstituted with a chimeric fusion protein comprising thioredoxin from Escherichia coli and the manganese stabilising protein from Thermosynechococcus elongatus. Surprisingly, even though E. coli thioredoxin is completely unrelated to PSII, the fusion protein restores higher rates of activity upon rebinding to PSII than either the native spinach MSP, or T. elongatus MSP. PSII reconstituted with the fusion protein also has a lower requirement for calcium than PSII with the small extrinsic proteins removed, or PSII reconstituted with spinach or T. elongatus MSP. The MSP portion of the fusion protein is less thermally stable compared to isolated MSP from T. elongatus, which could be the key to its superior activation capability through greater flexibility. This work reveals the importance of protein–protein interactions in the water splitting activity of PSII and suggests that conformational configurations, which increase flexibility in MSP, are essential to its function, even when these are induced by an unrelated protein.     

Is the manganese stabilizing 33 kDa protein of photosystem II attaining a 'natively unfolded' or 'molten globule' structure in solution?

This study compares the properties of the extrinsic 33 kDa subunit acting as 'manganese stabilizing protein' (MSP) of the water oxidizing complex with characteristic features of proteins that are known to attain a 'natively unfolded' or a 'molten globule' structure. The analysis leads to the conclusion that the MSP in solution is most likely a 'molten globule' with well defined compact regions of beta structure. The possible role of these structural peculiarities of MSP in solution for its function as important constituent of the WOC is discussed.     

Specific activity of α-l-fucosidase in sera with phenotypes of either low,intermediate, or high total enzyme activity and in a fucosidosis serum

The quantity of -l-fucosidase activity in human serum is determined by heredity. An individual may inherit either low, intermediate, or high serum enzyme activity. An enzyme-linked immunoabsorbent assay has been developed that can detect 0.3 ng of -l-fucosidase protein. Enzyme protein in serum of 102 individuals ranged from 20 to 835 ng/ml. The group included individuals with low, intermediate, and high enzyme activity. The specific activity of -l-fucosidase within this group was statistically the same (mean±SD=11,002±1051 U/mg). Thus, individuals with low and intermediate enzyme activity in serum had lower amounts of enzyme protein with the same specific activity as in individuals with high enzyme activity. Fucosidosis is a rare inherited disease in which -l-fucosidase activity in tissues and body fluids is low or absent. The concentrations of enzyme protein in sera of a fucosidosis patient and parents were 76, 565, and 604 ng/ml, respectively, and the specific activities of enzyme were 1316, 8938, and 8858 U/mg, respectively. Thus, the fucosidosis serum probably contained a structurally altered enzyme with reduced catalytic activity. The somewhat low specific activities in the parents suggested that their sera contained both structurally altered and normal protein.This research was supported by National Institutes of Health Grants AM 32161 and GM 31425.     

Mutagenicity of flavones,chromones and acetophenones in Salmonella typhimurium: New structure—activity relationships

28 flavones and 11 structurally-related flavonoids, chromones, and acetophenones, were tested for mutagenicity in the Salmonella typhimurium his reversion assay. 7 flavones, all of which were hydroxy- or methoxy-substituted at position 8, were moderate to strong mutagens in strain TA100 in the presence of rat liver S9 mix. In each case, the response of strain TA98 was either not significant or was very much weaker than that observed in strain TA100. The activation by S9 is not mediated by the microsomal cytochrome P₄₅₀ system, since activation was not diminished when microsomes were removed by centrifugation at 100000 × g. The observed strain specificity and structural requirements for activity indicate a mutagenic mechanism different from that associated with previously reported mutagenic flavonols (3-hydroxy-flavones) which are most active in strain TA98. The most mutagenic flavone investigated, 5,7,8-trihydroxy-flavone (norwogonin), had a potency of 17 revertants/nmole.Simplification of the chemical structures to hydroxy-substituted chromone and acetophenone systems revealed similar strain specificity, hydroxylation requirements, and S9 dependence within these structural classes, suggesting a similar activation pathway and mutagenic mechanism. The greatest mutagenic potency was observed within the flavone series, but significant potency was retained by similarly hydroxylated chromones and acetophenones. No mutagenic activity was observed in the absence of the aryl ketone moiety.     

Conformational folding and stability of the HET-C2 glycolipid transfer protein fold: does a molten globule-like state regulate activity?

The glycolipid transfer protein (GLTP) superfamily is defined by the human GLTP fold that represents a novel motif for lipid binding and transfer and for reversible interaction with membranes, i.e., peripheral amphitropic proteins. Despite limited sequence homology with human GLTP, we recently showed that HET-C2 GLTP of Podospora anserina is organized conformationally as a GLTP fold. Currently, insights into the folding stability and conformational states that regulate GLTP fold activity are almost nonexistent. To gain such insights into the disulfide-less GLTP fold, we investigated the effect of a change in pH on the fungal HET-C2 GLTP fold by taking advantage of its two tryptophans and four tyrosines (compared to three tryptophans and 10 tyrosines in human GLTP). pH-induced conformational alterations were determined by changes in (i) intrinsic tryptophan fluorescence (intensity, emission wavelength maximum, and anisotropy), (ii) circular dichroism over the near-UV and far-UV ranges, including thermal stability profiles of the derivatized molar ellipticity at 222 nm, (iii) fluorescence properties of 1-anilinonaphthalene-8-sulfonic acid, and (iv) glycolipid intermembrane transfer activity monitored by Fo?rster resonance energy transfer. Analyses of our recently determined crystallographic structure of HET-C2 (1.9 ?) allowed identification of side chain electrostatic interactions that contribute to HET-C2 GLTP fold stability and can be altered by a change in pH. Side chain interactions include numerous salt bridges and interchain cation-π interactions, but not intramolecular disulfide bridges. Histidine residues are especially important for stabilizing the local positioning of the two tryptophan residues and the conformation of adjacent chains. Induction of a low-pH-induced, molten globule-like state inhibited glycolipid intermembrane transfer by the HET-C2 GLTP fold.     

Polymorphism in the exon 4 of β-lactoglobulin variant B precursor gene and its association with milk traits and protein structure in Chinese Holstein

β-lactoglobulin (β-LG) is the major whey protein in the milk. In order to investigate the polymorphism of β-LG variant B precursor (β-LG B*: GenBank accession no. DQ489319) gene and its effects on the milk traits, the single-strand conformation polymorphism method (PCR-SSCP) were adopted to analyze polymorphism between 5229th and 5476th bp in the β-LG B* gene in Chinese Holstein. Four genotypes were found (AA, AB, AC and ABC) and 3 single nucleotide polymorphisms (SNPs) were detected (g.5239C>A, g.5240A>C, g.5305C>T and mix type g.5305C/T) in the exon 4 of β-LG B* gene. It was also found that the protein contents of AB, AC and ABC dairy cows were higher than AA (P < 0.05), and AC cows were the highest among them. Three SNPs (g.5239C>A, g.5240A>C and g.5305C>T) might affect the milk trait and all of them were high polymorphism (0.5 < PIC < 1.0). In further researches, the three SNPs also caused amino acid change (Asp>Glu, Thr>Pro and Ala>Val) respectively, and the spatial secondary and tertiary structure forecasting result also showed that single amino acid change influence protein spatial structure change in Chinese Holstein. Taken together, it is suggested that these SNPs change β-LG B* gene structure and expression. The polymorphism possibly holds the secret of milk protein and fat contents in the milk of Chinese Holstein.     

Synthesis, pharmacological activity and structure affinity relationships of spirocyclic σ(1) receptor ligands with a (2-fluoroethyl) residue in 3-position

In order to develop a fluorinated radiotracer for imaging of ??₁ receptors in the central nervous system a series of (2-fluoroethyl) substituted spirocyclic piperidines 3 has been prepared. In the key step of the synthesis 2-bromocinnamaldehyde acetal 5 was added to piperidones 6 with various substituents at the N-atom. Unexpectedly, this reaction led to 2-benzoxepines 8, which were contracted with acid to afford the spirocyclic 2-benzofuranacetaldehydes 9. The best yields were obtained, when the transformations up to the alcohols 10 were performed without isolation of intermediates. Generally the (2-fluoroethyl) derivatives 3 have higher ??₁ affinity and ??₁/??₂ selectivity than the corresponding (3-fluoropropyl) derivatives 2. The most promising candidate for the development as radiotracer is the (2-fluoroethyl) derivative 3a (WMS-1828, fluspidine, 1′-benzyl-3-(2-fluoroethyl)-3H-spiro[[2]benzofuran-1,4′-piperidine]), which shows subnanomolar ??₁ affinity (K_i = 0.59 nM) and excellent selectivity over the ??₂ subtype (1331-fold) as well as some other receptor systems. The novel synthetic strategy also allows the systematic pharmacological evaluation of intermediate alcohols 10. Despite their high ??₁ affinity (K_i = 6-32 nM) and selectivity the alcohols 10 are 10-30-fold less potent than the bioisosteric fluoro derivatives 3.     

Native vegetation structure and persistence of endangered Tehuantepec jackrabbits in a neotropical savanna in Oaxaca, México

Because of its crucial importance for the persistence of the endangered Tehuantepec jackrabbit (Lepus flavigularis), we identified structural characteristics of native vegetation (1) selected by Tehuantepec jackrabbits to establish home ranges, (2) used within home ranges, and (3) in relation to activity and inactivity periods. A neotropical savanna in the Isthmus of Tehuantepec, Oaxaca, México, offered diversity in native flora and heterogeneity in vegetation structure to the studied population of Tehuantepec jackrabbit. Adults and juveniles differed in habitat use patterns. No effect on habitat selection was detected for sex or season. Jackrabbits established home ranges on grassy habitat with discontinuous overstory of nanche (Byrsonima crassifolia) shrubbery and morro (Crescentia) trees. Adults underused dense vegetation where predators may ambush them; juveniles avoided dense vegetation and grassland without overstory. Within adults’ home ranges, habitat selection favoring grassy habitats with nanche and morro was relatively stronger during the activity period (nocturnal and crepuscular hours) and during one of 2 years which had more rainfall. Scattered trees and open shrubbery likely allowed foraging jackrabbits with both visibility and escape routes used to detect and outrun predators. Moreover, during the inactivity period (diurnal hours) adults and juveniles favored nanche shrubbery that provided resting jackrabbits with shelter to hide from predators. Clearly, structure and diversity of native vegetation in the savanna needs to be preserved for conservation of Tehuantepec jackrabbits because deteriorated habitat may pose higher predation rates for a jackrabbit population in risk of extirpation.     

The biology of desmin filaments: how do mutations affect their structure, assembly, and organisation?

Desmin, the major intermediate filament (IF) protein of muscle, is evolutionarily highly conserved from shark to man. Recently, an increasing number of mutations of the desmin gene has been described to be associated with human diseases such as certain skeletal and cardiac myopathies. These diseases are histologically characterised by intracellular aggregates containing desmin and various associated proteins. Although there is progress regarding our knowledge on the cellular function of desmin within the cytoskeleton, the impact of each distinct mutation is currently not understood at all. In order to get insight into how such mutations affect filament assembly and their integration into the cytoskeleton we need to establish IF structure at atomic detail. Recent progress in determining the dimer structure of the desmin-related IF-protein vimentin allows us to assess how such mutations may affect desmin filament architecture.     

An inhibitory effect of RGD peptide on protein, priming reaction of bacteriophages ?29 and M2

Summary The amino acid sequence, arginine-glycine-aspartic acid (RGD), found in some cell adhesive proteins, is a recognition signal for the receptor protein. It is interesting that we have found the RGD sequence in terminal protein (TP) of bacteriophages 29 and M2 near an amino acid, the serine residue at 232, covalently linked to the terminal nucleotide of their DNAs. At the initiation of proteinprimed DNA replication, TP is essential for the recognition of replication machinery containing DNA polymerase and primer protein (PP; PP becomes TP upon linking the first nucleotide, and hence the primary structure of TP is the same as that of PP). Synthetic peptide RGD specifically inhibited transfection of 29 and M2. The target of the RGD peptide is shown to be TP by marker rescue experiments, suggesting that a receptor for the RGD sequence exists in TP. Furthermore, the peptide inhibited the in vitro protein-priming reaction of DNA replication. We propose that the RGD sequence of PP and a putative receptor on TP is utilized for the molecular recognition initiating DNA replication.