Structural characterization of the N-terminal autoregulatory sequence of phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine, and through phosphorylation by cAMP-dependent protein kinase at Ser 16 in the N-terminal autoregulatory sequence of the enzyme. The crystal structures of phosphorylated and unphosphorylated forms of the enzyme showed that, in the absence of phenylalanine, in both cases the N-terminal 18 residues including the phosphorylation site contained no interpretable electron density. We used nuclear magnetic resonance (NMR) spectroscopy to characterize this N-terminal region of the molecule in different stages of the regulatory pathway. A number of sharp resonances are observed in PAH with an intact N-terminal region, but no sharp resonances are present in a truncation mutant lacking the N-terminal 29 residues. The N-terminal sequence therefore represents a mobile flexible region of the molecule. The resonances become weaker after the addition of phenylalanine, indicating a loss of mobility. The peptides corresponding to residues 2-20 of PAH have different structural characteristics in the phosphorylated and unphosphorylated forms, with the former showing increased secondary structure. Our results support the model whereby upon phenylalanine binding, the mobile N-terminal 18 residues of PAH associate with the folded core of the molecule; phosphorylation may facilitate this interaction.

Synthesis and structural properties of patellamide a derivatives and their copper(II) compounds

The synthesis, characterization and copper(II) coordination chemistry of three new cyclic peptide ligands, PatJ(1) (cyclo-(Ile -Thr- (Gly)Thz-lle-Thr(Gly)Thz)), PatJ(2) (cyclo-(Ile-Thr(Gly)Thz-(D)-Ile-Thr-(Gly)Thz)), and PatL (cyclo-(Ile-Ser-(Gly)Thz-Ile-Ser(Gly)Thz)) are reported. All of these cyclic peptides and PatN (cyclo-(Ile-Ser(Gly)Thz-Ile-Thr-(Gly)Thz)) are derivatives of patellamide A and have a [24]azacrown-8 macrocyclic structure. All four synthetic cyclic peptides have two thiazole rings but, in contrast to patellamide A, no oxazoline rings. The molecular structure of PatJ1, determined by X-ray crystallography, has a saddle conformation with two close-to-co-parallel thiazole rings, very similar to the geometry of patellamide D. The two coordination sites of PatJ1 with thiazole-N and amide-N donors are each well preorganized for transition metal ion binding. The coordination of copper(II) was monitored by UV/Vis spectroscopy, and this reveals various (meta)stable mono- and dinuclear copper(II) complexes whose stoichiometry was confirmed by mass spectra. Two types of dinuclear copper(II) complexes, [Cu-2(H4L)(OH2)(n)](2+) (n = 6, 8) and [Cu-2(H4L)(OH2)(n)] (n=4, 6; L=PatN, PatL, PatJ1, PatJ2) have been identified and analyzed structurally by EPR spectroscopy and a combination of spectra simulations and molecular mechanics calculations (MM-EPR). The four structures are similar to each other and have a saddle conformation, that is, derived from the crystal structure of PatJ(1) by a twist of the two thiozole rings. The small but significant structural differences are characterized by the EPR simulations.

Comparative influence of forest management and habitat structural factors on the abundances of hollow-nesting bird species in subtropical Australian eucalypt forest

We examined the impact of single-tree selective logging and fuel reduction bums on the abundance of hollow-nesting bird species at a regional scale in southeastern Queensland, Australia. Data were collected on species abundance and habitat structure of dry sclerophyll production forest at 36 sites with known logging and fire histories. Sixteen bird species were recorded with most being resident, territorial, obligate hollow nesters that used hollows that were either small (18 cm diameter). Species densities were typically low, but combinations of two forest management and three habitat structural variables influenced the abundances of eight bird species in different and sometimes conflicting ways. The results suggest that habitat tree management for biodiversity in production forests cannot depend upon habitat structural characteristics alone. Management histories appear to have independent influence (on some bird species) that are distinguishable from their impacts on habitat structure per se. Rather than managing to maximize species abundances to maintain biodiversity, we may be better off managing to avoid extinctions of populations by identifying thresholds of acceptable fluctuations in populations of not only hollow-nesting birds but other forest dependent wildlife relative to scientifically valid forest management and habitat structural surrogates.

Reversible luminescence thermochromism in dipotassiumsodium tris[dicyanoargentate(I)] and the role of structural phase transitions

As a function of temperature, the layered compound K2Na[Ag(CN)213 displays dramatic variations in luminescence thermochromism with major trend changes occurring around 80 K. In order to understand these interesting optical properties, high-resolution neutron diffraction investigations were performed on a polycrystalline sample of this material in the temperature range from 1.5 to 300 K, and previous synchrotron X-ray data of Larochelle et al. (Solid State Commun. 114, 155 (2000)) were reinterpreted. The corresponding significant structural changes were found to be continuous with an anomalous increase of the monoclinic c-lattice parameter with decreasing temperature, associated with slight reorientations of two inequivalent, approximately linear N-C-Ag-C-N units. In the whole temperature range, the crystal structure is monoclinic with the space group C2/m. Based on the structural results, the major luminescence thermochromism changes around 80 K are attributed to the dominance of a back energy transfer process from low- to high-energy excitons at high temperatures. (E) 2002 Elsevier Science (USA).

Copolymers obtained by the radiation-induced grafting of styrene onto poly(tetrafluoroethylene-co-perfluoropropylvinyl ether) substrates. 1. Preparation and structural investigation

A study has been made to investigate the radiation grafting of styrene onto poly(tetrafluoroethylene-co-perfluoropropylvinyl ether) (PFA) substrates, using the simultaneous irradiation method. Two PFA polymers of different comonomer perfluoropropyl vinyl ether (PPVE) content and degree of crystallinity were used. Effects of grafting conditions such as monomer concentrations, type of solvent, dose rate, and irradiation dose on the grafting yield were investigated. Of the six different solvents used, the most efficient in terms of increasing grafting yield were dichloromethane, benzene, and methanol. The degree of grafting increased with increasing radiation dose up to 500 kGy, stabilizing above this dose. However, the grafting yield decreased with an increase in the dose rate. The grafting of styrene onto the PFA substrates was confirmed by FTIR-ATR and micro-Raman spectroscopy, The increase in the overall grafting yield was accompanied by a proportional increase in the penetration depth of the grafts into the substrate.

Differential regulation of twitching motility and elastase production by Vfr in Pseudomonas aeruginosa

Vfr, a homolog of Escherichia coli cyclic AMP (cAMP) receptor protein, has been shown to regulate quorum sensing, exotoxin A production, and regA transcription in Pseudomonas aeruginosa. We identified a twitching motility-defective mutant that carries a transposon insertion in vfr and confirmed that vfr is required for twitching motility by construction of an independent allelic deletion-replacement mutant of vfr that exhibited the same phenotype, as well as by the restoration of normal twitching motility by complementation of these mutants with wild-type vfr. Vfr-null mutants exhibited severely reduced twitching motility with barely detectable levels of type IV pili, as well as loss of elastase production and altered pyocyanin production. We also identified reduced-twitching variants of quorum-sensing mutants (PAK lasl::Tc) with a spontaneous deletion in vfr (S. A. Beatson, C. B. Whitchurch, A. B. T. Semmler, and J. S. Mattick, J. Bacteriol., 184:3598-3604,2002), the net result of which was the loss of five residues (EQERS) from the putative cAMP-binding pocket or Vfr. This allele (VfrDeltaEQERS) was capable of restoring elastase and pyocyanin production to wild-type levels in vfr-null mutants but not their defects in twitching motility. Furthermore, structural analysis of Vfr and VfrDeltaEQERS in relation to E. coli CRP suggests that Vfr is capable of binding both cAMP and cyclic GMP whereas VfrDeltaEQERS is only capable of responding to cAMP. We suggest that Vfr controls twitching motility and quorum sensing via independent pathways in response to these different signals, bound by the same cyclic nucleotide monophosphate-binding pocket.

Negative ion electrospray mass spectra of caerulein peptides: an aid to structural determination

MS/MS data derived from the [M-H](-) ions of desulfated caerulein peptides provide (i) sequencing information from a combination of alpha, beta and gamma backbone cleavages, and (ii) identification of specific amino acid side chains by side-chain cleavages [e.g. Ser (-CH2O), Thr (-CH3CHO) and Asp (-H2O)] (fragmentations having no counterparts in positive ion spectra). In addition, delta and/or gamma backbone cleavage ions from Asp residues identify the position of these residues in the peptide. In contrast, neither delta nor gamma cleavage ions are observed from either the Gln2 residue nor from Phe residues. Full structural information can be obtained from a consideration of the positive and negative ion MS/MS data in concert. Copyright (C) 2002 John Wiley Sons, Ltd.

Structural changes in mixed Langmuir-Blodgett films upon nanoparticle formation

The formation of CdS nanoparticles by reacting mixed Langmuir-Blodgett films of arachidic acid and either octadecylamine or dimethyldioctadecylammonium nitrate on a cadmium-containing subphase with hydrogen sulfide gas has resulted in the identification of a number of structural changes, observed using grazing incidence X-ray diffraction. In the case of octadecylamine, the structure after reaction is a hexagonal close-packed array of surfactant-stabilized nanoclusters, with a lattice constant of a = 17.65 Angstrom. In both octadecylamine and dimethyldioctadecylammonium nitrate films, the presence of a unit cell tilted at 38degrees to the plane of the substrate was found. Despite these changes, the average nanoparticle size was unaffected by the addition of either second component to the film.

Structural and surface property changes of macadamia nut-shell char upon activation and high temperature treatment

Structural and surface property changes of macadamia nut-shell (MNS) char upon activation and high temperature treatment (HTT) were studied by high-resolution nitrogen adsorption, diffuse reflectance infra-red Fourier transform spectroscopy, X-ray photoelectron spectroscopy, and temperature-programmed desorption. It is found that activation of MNS char can be divided into the low extent activation which may involve the reactions of internal oxygen-containing groups and leads to the formation of comparatively uniform micropores, and the high extent activation which induces reactions between carbon and activating gas and produces a large amount of micropores. The surface functional groups (SFGs) basically increase with the increase of activation extent, but high extent activation preferentially increases the amount of -C-O and -C=O. HTT in air for a short tithe at a high temperature (1173 K) greatly increases the micropore volume and the amounts of SFGs. By appropriately choosing the activation and HTT conditions, it is possible to control both the textural structure and the type and amounts of SFG. (C) 2002 Published by Elsevier Science Ltd.

Effect of a control burn on lizards and their structural environment in a eucalypt open-forest

The composition of an open-forest lizard assemblage in eastern Australia was examined before and after a low-intensity controlled fire and concurrently compared with that in an adjoining unburnt area. The effect of fire on the available structural environment and the habitat used by two focal species, Carlia vivax and Lygisaurus foliorum, was also examined. Lizard species richness was unaffected by the controlled burn as was the abundance of most species. C. vivax was the only species to display a significant reduction in abundance after fire. While the low-intensity fire resulted in significant changes to the available structural environment, there were no compensatory shifts in the habitat preferences of either C. vivax or L. foliorum. The reduction in abundance of C. vivax was congruent with this species' avoidance of burnt areas. C. vivax displayed a non-random preference for ground cover and litter cover, which were reduced in burnt areas. Changes in the availability of preferred structural habitat features are likely to contribute to changes in the abundance of some lizard species. Therefore, even low-intensity disturbances can have an impact on lizard assemblages if critical habitat features are lost or become limiting.

Characterization of the retinoid orphan-related receptor-alpha coactivator binding interface: A structural basis for ligand-independent transcription

The retinoid orphan-related receptor-alpha (RORalpha) is a member of the ROR subfamily of orphan receptors and acts as a constitutive activator of transcription in the absence of exogenous ligands. To understand the basis of this activity, we constructed a homology model of Rill using the closely related TRbeta as a template. Molecular modeling suggested that bulky hydrophobic side chains occupy the RORa ligand cavity leaving a small but distinct cavity that may be involved in receptor stabilization. This model was subject to docking simulation with a receptor-interacting peptide from the steroid receptor coactivator, GR-interacting protein-1, which delineated a coactivator binding surface consisting of the signature motif spanning helices 3-5 and helix 12 [activation function 2 (AF2)]. Probing this surface with scanning alanine mutagenesis showed structural and functional equivalence between homologous residues of RORalpha and TRbeta. This was surprising (given that Rill is a ligand-independent activator, whereas TRbeta has an absolute requirement for ligand) and prompted us to use molecular modeling to identify differences between Rill and TRbeta in the way that the All helix interacts with the rest of the receptor. Modeling highlighted a nonconserved amino acid in helix 11 of RORa (Phe491) and a short-length of 3.10 helix at the N terminus of AF2 which we suggest i) ensures that AF2 is locked permanently in the holoconformation described for other liganded receptors and thus 2) enables ligand-independent recruitment of coactivators. Consistent with this, mutation of RORa Phe491 to either methionine or alanine (methionine is the homologous residue in TRbeta), reduced and ablated transcriptional activation and recruitment of coactivators, respectively. Furthermore, we were able to reconstitute transcriptional activity for both a deletion mutant of Ill lacking All and Phe491 Met, by overexpression of a GAL-AF2 fusion protein, demonstrating ligand-independent recruitment of AF2 and a role for Phe491 in recruiting AF2.

Structural ordering of coal char during heat treatment and its impact on reactivity

The effect of heat treatment on the structure of an Australian semi-anthracite char was studied in detail in the 850-1150degreesC temperature range using XRD, HRTEM, and electrical resistivity techniques. It was found that the carbon crystallite size in the char does not change significantly during heat treatment in the temperature range studied, for both the raw coal and its ash-free derivative obtained by acid treatment. However, the fraction of the organized carbon in the raw coal chars, determined by XRD, increased with increase of heat treatment time and temperature, while that for the ash-free coal chars remained almost unchanged. This suggests the occurrence of catalytic ordering during heat treatment, supported by the observation that the electrical resistivity of the raw coal chars decreased with heat treatment, while that of the ash-free coal chars did not vary significantly. Further confirmatory evidence was provided by high resolution transmission electron micrographs depicting well-organized carbon layers surrounding iron particles. It is also found that the fraction of organized carbon does not reach unity, but attains an apparent equilibrium value that increases with increase in temperature, providing an apparent heat of ordering of 71.7 kJ mol(-1) in the temperature range studied. Good temperature-independent correlation was found between the electrical resistivity and the organized carbon fraction, indicating that electrical resistivity is indeed structure sensitive. Good correlation was also found between the electrical resistivity and the reactivity of coal char. All these results strongly suggest that the thermal deactivation is the result of a crystallite-perfecting process, which is effectively catalyzed by the inorganic matter in the coal char. Based on kinetic interpretation of the data it is concluded that the process is diffusion controlled, most likely involving transport of iron in the inter-crystallite nanospaces in the temperature range studied. The activation energy of this transport process is found to be very low, at about 11.8 kJ mol(-1), which is corroborated by model-free correlation of the temporal variation of organized carbon fraction as well as electrical resistivity data using the superposition method, and is suggestive of surface transport of iron. (C) 2002 Elsevier Science Ltd. All rights reserved.

A new level of conotoxin diversity, a non-native disulfide bond connectivity in alpha-conotoxin AuIB reduces structural definition but increases biological activity

alpha-Conotoxin AuIB and a disulfide bond variant of AuIB have been synthesized to determine the role of disulfide bond connectivity on structure and activity. Both of these peptides contain the 15 amino acid sequence GCCSYPPCFATNPDC, with the globular (native) isomer having the disulfide connectivity Cys(2-8 and 3-15) and the ribbon isomer having the disulfide connectivity Cys(2-15 and 3-8). The solution structures of the peptides were determined by NAIR spectroscopy, and their ability to block the nicotinic acetylcholine receptors on dissociated neurons of the rat parasympathetic ganglia was examined. The ribbon disulfide isomer, although having a less well defined structure, is surprisingly found to have approximately 10 times greater potency than the native peptide. To our knowledge this is the first demonstration of a non-native disulfide bond isomer of a conotoxin exhibiting greater biological activity than the native isomer.

The structural and functional diversity of naturally occurring antimicrobial peptides

Antimicrobial peptides occur in a diverse range of organisms from microorganisms to insects, plants and animals. Although they all have the common function of inhibiting or killing invading microorganisms they achieve this function using an extremely diverse range of structural motifs. Their sizes range from approximately 10-90 amino acids. Most carry an overall positive charge, reflecting a preferred mode of electrostatic interaction with negatively charged microbial membranes. This article describes the structural diversity of a representative set of antimicrobial peptides divided into five structural classes: those with agr-helical structure, those with bgr-sheet structure, those with mixed helical / bgr- sheet structure, those with irregular structure, and those incorporating a macrocyclic structure. There is a significant diversity in both the size and charge of molecules within each of these classes and between the classes. The common feature of their three-dimensional structures is, however, that they have a degree of amphipathic character in which there is separate localisation of hydrophobic regions and positively charged regions. An emerging trend amongst antimicrobial proteins is the discovery of more macrocyclic analogues. Cyclisation appears to impart an additional degree of stability on these molecules and minimizes proteolytic cleavage. In conclusion, there appear to be a number of promising opportunities for the development of novel clinically useful antimicrobial peptides based on knowledge of the structures of naturally occurring antimicrobial molecules.

Synthesis and structural analysis of the N-terminal domain of the thyroid hormone-binding protein transthyretin

Transthyretin (TTR) is a 55 kDa protein responsible for the transport of thyroid hormones and retinol in human serum. Misfolded forms of the protein are implicated in the amyloid diseases familial amyloidotic polyneuropathy and senile systemic amyloidosis. Its folding properties and stabilization by ligands are of current interest due to their importance in understanding and combating these diseases. To assist in such studies we developed a method for the solid phase synthesis of the monomeric unit of a TTR analogue and its folding to form a functional 55 kDa tetramer. The monomeric unit of the protein was chemically synthesized in three parts, comprising amino acid residues 151, 5499 and 102127, and ligated using chemoselective thioether ligation chemistry. The synthetic protein was folded and assembled to a tetrameric structure in the presence of the TTRs native ligand, thyroxine, as shown by gel filtration chromatography, native gel electrophoresis, TTR antibody recognition and thyroid hormone binding. In the current study the solution structure of the first of these fragment peptides, TTR(151) is examined to determine its intrinsic propensity to form beta-sheet structure, potentially involved in amyloid fibril formation by TTR. Despite the presence of extensive beta-structure in the native form of the protein, the Nterminal fragment adopts an essentially random coil conformation in solution.