Conformational profiling of a G-rich sequence within the c-KIT promoter

G-quadruplexes (G4) within oncogene promoters are considered to be promising anticancer targets. However, often they undergo complex structural rearrangements that preclude a precise description of the optimal target. Moreover, even when solved structures are available, they refer to the thermodynamically stable forms but little or no information is supplied about their complex multistep folding pathway. To shed light on this issue, we systematically followed the kinetic behavior of a G-rich sequence located within the c-KIT proximal promoter (kit2) in the presence of monovalent cations K + and Na + . A very short-lived intermediate was observed to start the G4 folding process in both salt conditions. Subsequently, the two pathways diverge to produce distinct thermodynamically stable species (parallel and antiparallel G-quadruplex in K + and Na + , respectively). Remarkably, in K + -containing solution a branched pathway is required to drive the wild type sequence to distribute between a monomeric and dimeric G-quadruplex. Our approach has allowed us to identify transient forms whose relative abundance is regulated by the environment; some of them were characterized by a half-life within the timescale of physiological DNA processing events and thus may represent possible unexpected targets for ligands recognition

Structural insights into Clostridium perfringens delta toxin pore formation

Clostridium perfringens Delta toxin is one of the three hemolysin-like proteins produced by C. perfringens type C and possibly type B strains. One of the others, NetB, has been shown to be the major cause of Avian Nectrotic Enteritis, which following the reduction in use of antibiotics as growth promoters, has become an emerging disease of industrial poultry. Delta toxin itself is cytotoxic to the wide range of human and animal macrophages and platelets that present GM2 ganglioside on their membranes. It has sequence similarity with Staphylococcus aureus β-pore forming toxins and is expected to heptamerize and form pores in the lipid bilayer of host cell membranes. Nevertheless, its exact mode of action remains undetermined. Here we report the 2.4 Å crystal structure of monomeric Delta toxin. The superposition of this structure with the structure of the phospholipid-bound F component of S. aureus leucocidin (LukF) revealed that the glycerol molecules bound to Delta toxin and the phospholipids in LukF are accommodated in the same hydrophobic clefts, corresponding to where the toxin is expected to latch onto the membrane, though the binding sites show significant differences. From structure-based sequence alignment with the known structure of staphylococcal α-hemolysin, a model of the Delta toxin pore form has been built. Using electron microscopy, we have validated our model and characterized the Delta toxin pore on liposomes. These results highlight both similarities and differences in the mechanism of Delta toxin (and by extension NetB) cytotoxicity from that of the staphylococcal pore-forming toxins

Crystal structure of monomeric human β-2- microglobulin reveals clues to its amyloidogenic properties

Dissociation of human β-2-microglobulin (β(2)m) from the heavy chain of the class I HLA complex is a critical first step in the formation of amyloid fibrils from this protein. As a consequence of renal failure, the concentration of circulating monomeric β(2)m increases, ultimately leading to deposition of the protein into amyloid fibrils and development of the disorder, dialysis-related amyloidosis. Here we present the crystal structure of a monomeric form of human β(2)m determined at 1.8-Å resolution that reveals remarkable structural changes relative to the HLA-bound protein. These involve the restructuring of a β bulge that separates two short β strands to form a new six-residue β strand at one edge of this β sandwich protein. These structural changes remove key features proposed to have evolved to protect β sheet proteins from aggregation [Richardson, J.&Richardson, D. (2002) Proc. Natl. Acad. Sci. USA 99, 2754–2759] and replaces them with an aggregationcompetent surface. In combination with solution studies using (1)H NMR, we show that the crystal structure presented here represents a rare species in solution that could provide important clues about the mechanism of amyloid formation from the normally highly soluble native protein

Conformational Transitions Accompanying Oligomerization of Yeast Alcohol Oxidase, a Peroxisomal Flavoenzyme

Alcohol oxidase (AO) is a homo-octameric flavoenzyme which catalyzes methanol oxidation in methylotrophic yeasts. AO protein is synthesized in the cytosol and subsequently sorted to peroxisomes where the active enzyme is formed. To gain further insight in the molecular mechanisms involved in AO activation, we studied spectroscopically native AO from Hansenula polymorpha and Pichia pastoris and three putative assembly intermediates. Fluorescence studies revealed that both Trp and FAD are suitable intramolecular markers of the conformation and oligomeric state of AO. A direct relationship between dissociation of AO octamers and increase in Trp fluorescence quantum yield and average fluorescence lifetime was found. The time-resolved fluorescence of the FAD cofactor showed a rapid decay component which reflects dynamic quenching due to the presence of aromatic amino acids in the FAD-binding pocket. The analysis of FAD fluorescence lifetime profiles showed a remarkable resemblance of pattern for purified AO and AO present in intact yeast cells. Native AO contains a high content of ordered secondary structure which was reduced upon FAD-removal. Dissociation of octamers into monomers resulted in a conversion of β-sheets into α-helices. Our results are explained in relation to a 3D model of AO, which was built based on the crystallographic data of the homologous enzyme glucose oxidase from Aspergillus niger. The implications of our results for the current model of the in vivo AO assembly pathway are discussed.

Liquid crystal phase and waterlike anomalies in a core-softened shoulder-dumbbells system

Using molecular dynamics we investigate the thermodynamics, dynamics and structure of 250 diatomic molecules interacting by a core-softened potential. This system exhibits thermodynamics, dynamics and structural anomalies: a maximum in density-temperature plane at constante pressure and maximum and minimum points in the diffusivity and translational order parameter against density at constant temperature. Starting with very dense systems and decreasing density the mobility at low temperatures first increases, reach a maximum, then decreases, reach a minimum and finally increases. In the pressure-temperature phase diagram the line of maximum translational order parameter is located outside the line of diffusivity extrema that is enclosing the temperature of maximum density line. We compare our results with the monomeric system showing that the anisotropy due to the dumbbell leads to a much larger solid phase and to the appearance of a liquid crystal phase. the double ranged thermodynamic and dynamic anomalies.Comment: 14 pages, 5 figure

A transient homotypic interaction model for the influenza A virus NS1 protein effector domain

Influenza A virus NS1 protein is a multifunctional virulence factor consisting of an RNA binding domain (RBD), a short linker, an effector domain (ED), and a C-terminal 'tail'. Although poorly understood, NS1 multimerization may autoregulate its actions. While RBD dimerization seems functionally conserved, two possible apo ED dimers have been proposed (helix-helix and strand-strand). Here, we analyze all available RBD, ED, and full-length NS1 structures, including four novel crystal structures obtained using EDs from divergent human and avian viruses, as well as two forms of a monomeric ED mutant. The data reveal the helix-helix interface as the only strictly conserved ED homodimeric contact. Furthermore, a mutant NS1 unable to form the helix-helix dimer is compromised in its ability to bind dsRNA efficiently, implying that ED multimerization influences RBD activity. Our bioinformatical work also suggests that the helix-helix interface is variable and transient, thereby allowing two ED monomers to twist relative to one another and possibly separate. In this regard, we found a mAb that recognizes NS1 via a residue completely buried within the ED helix-helix interface, and which may help highlight potential different conformational populations of NS1 (putatively termed 'helix-closed' and 'helix-open') in virus-infected cells. 'Helix-closed' conformations appear to enhance dsRNA binding, and 'helix-open' conformations allow otherwise inaccessible interactions with host factors. Our data support a new model of NS1 regulation in which the RBD remains dimeric throughout infection, while the ED switches between several quaternary states in order to expand its functional space. Such a concept may be applicable to other small multifunctional proteins

Modeling the hydrolysis of perfluorinated compounds containing carboxylic and phosphoric acid ester functions, alkyl iodides, and sulfonamide groups

Temperature dependent rate constants were estimated for the acid- and base-catalyzed and neutral hydrolysis reactions of perfluorinated telomer acrylates (FTAcrs) and phosphate esters (FTPEs), and the SN1 and SN2 hydrolysis reactions of fluorotelomer iodides (FTIs). Under some environmental conditions, hydrolysis of monomeric FTAcrs could be rapid (half-lives of several years in marine systems and as low as several days in some landfills) and represent a dominant portion of their overall degradation. Abiotic hydrolysis of monomeric FTAcrs may be a significant contributor to current environmental loadings of fluorotelomer alcohols (FTOHs) and perfluoroalkyl carboxylic acids (PFCAs). Polymeric FTAcrs are expected to be hydrolyzed more slowly, with estimated half-lives in soil and natural waters ranging between several centuries to several millenia absent additional surface area limitations on reactivity. Poor agreement was found between the limited experimental data on FTPE hydrolysis and computational estimates, requiring more detailed experimental data before any further modeling can occur on these compounds or their perfluoroalkyl sulfonamidoethanol phosphate ester (PFSamPE) analogs. FTIs are expected to have hydrolytic half-lives of about 130 days in most natural waters, suggesting they may be contributing to substantial FTOH and PFCA inputs in aquatic systems. Perfluoroalkyl sulfonamides (PFSams) appear unlikely to undergo abiotic hydrolysis at the S-N, C-S, or N-C linkages under environmentally relevant conditions, although potentially facile S-N hydrolysis via intramolecular catalysis by ethanol and acetic acid amide substituents warrants further investigation

Self-Templated Nucleation in Peptide and Protein aggregation

Peptides and proteins exhibit a common tendency to assemble into highly ordered fibrillar aggregates, whose formation proceeds in a nucleation-dependent manner that is often preceded by the formation of disordered oligomeric assemblies. This process has received much attention because disordered oligomeric aggregates have been associated with neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. Here we describe a self-templated nucleation mechanism that determines the transition between the initial condensation of polypeptide chains into disordered assemblies and their reordering into fibrillar structures. The results that we present show that at the molecular level this transition is due to the ability of polypeptide chains to reorder within oligomers into fibrillar assemblies whose surfaces act as templates that stabilise the disordered assemblies.Comment: 4 pages, 3 figure

Pivalolactone, 2. Copolyester synthesis via interchange reactions with polypivalolactone

The synthesis of copolyesters via interchange reactions of polypivalolactone (PPVL) with several compounds was studied. The synthetical procedures are two-stage melt processes: in the first stage ester bonds in the polymer chain are cleaved and new groups are incorporated in the polymer chain, while in the second step condensation of the end-groups formed occurs. For the synthesis of copolymers, three procedures were used, with tetrabutyl orthotitanate as a catalyst. PPVL was heated with equimolar mixtures of bisphenol-A diacetate (BPAac) and terephthalic acid (TA), but no copolymers were formed; instead, polycondensation of BPAac with TA occurred, leaving the PPVL unaffected. From PPVL and mixtures of BPAac and dimethyl terephthalate (DMT) polymers were obtained which contained a significant amount of copolymeric sequences. However, most of the polymeric chains consisted of PPVL and poly(bisphenol-A terephthalate) blocks. Random copolymers with thermal stability were obtained after heating PPVL with bisphenol-A polycarbonate and DMT. The latter process was studied in detail by IR, DSC, and solubility and selective degradation tests. Based on the results of these studies, the reactions occurring during the three procedures were discussed