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Understanding the crystallization behavior of semi-crystalline polymers during processing is key to achieve the desired material performances. Isotactic polypropylene characteristics are highly tunable and easy to adjust by changing the processing conditions thanks to its structural versatility and polymorphic behavior. Hereby, we performed for the first time ever a challenging real-time X-ray characterization of isotactic polypropylene during the cast film extrusion process by interfacing a compact cast film extruder machine with a synchrotron line. We report data of excellent quality recorded from the contact point between the just extruded polymer melt and the water-refrigerated cooling roll further down to the cooling roll line by using a grazing incidence scattering geometry. The explored cooling roll temperatures (from 70 °C to 15 °C) allowed to produce iPP films with defined structural composition, from α to mesophase. Evolution of the phase crystallinity as a function of the contact time between the polymer melt and the cooling roll clearly shows the presence of three crystallization zones: 1) an amorphous zone, 2) a frostline zone and lastly 3) a high-crystallinity zone. In the end, the experimental data have been matched with simulated data using a simple crystallization kinetics model, finding a good agreement in terms of crystalline development over the investigated time scale. As a closing remark, we hope that our effort will spark new interest in the real-time characterization of semi-crystalline materials and that will ultimately be utilized as a starting point to optimize and expand the characterization of these extremely important materials.

Devices made using thin-film semiconductors have attracted much interest recently owing to new application possibilities. Among materials systems suitable for thin-film electronics, organic semiconductors are of particular interest; their low cost, biocompatible carbon-based materials and deposition by simple techniques such as evaporation or printing enable organic semiconductor devices to be used for ubiquitous electronics, such as those used on or in the human body or on clothing and packages1–3. The potential of organic electronics can be leveraged only if the performance of organic transistors is improved markedly. Here we present organic bipolar transistors with outstanding device performance: a previously undescribed vertical architecture and highly crystalline organic rubrene thin films yield devices with high differential amplification (more than 100) and superior high-frequency performance over conventional devices. These bipolar transistors also give insight into the minority carrier diffusion length—a key parameter in organic semiconductors. Our results open the door to new device concepts of high-performance organic electronics with ever faster switching speeds.

We investigate the thermal behavior and a newly found high temperature polymorph of C10-DNTT thin films experimentally and theoretically.

The signaling molecule auxin coordinates many growth and development processes in plants, mainly through modulating gene expression. Transcriptional response is mediated by the family of auxin response factors (ARF). Monomers of this family recognize a DNA motif and can homodimerize through their DNA-binding domain (DBD), enabling cooperative binding to an inverted binding site. Most ARFs further contain a C-terminal PB1 domain that is capable of homotypic interactions and mediating interactions with Aux/IAA repressors. Given the dual role of the PB1 domain, and the ability of both DBD and PB1 domain to mediate dimerization, a key question is how these domains contribute to DNA-binding specificity and affinity. So far, ARF–ARF and ARF–DNA interactions have mostly been approached using qualitative methods that do not provide a quantitative and dynamic view on the binding equilibria. Here, we utilize a DNA binding assay based on single-molecule Förster resonance energy transfer (smFRET) to study the affinity and kinetics of the interaction of several Arabidopsis thaliana ARFs with an IR7 auxin-responsive element (AuxRE). We show that both DBD and PB1 domains of AtARF2 contribute toward DNA binding, and we identify ARF dimer stability as a key parameter in defining binding affinity and kinetics across AtARFs. Lastly, we derived an analytical solution for a four-state cyclic model that explains both the kinetics and the affinity of the interaction between AtARF2 and IR7. Our work demonstrates that the affinity of ARFs toward composite DNA response elements is defined by dimerization equilibrium, identifying this as a key element in ARF-mediated transcriptional activity.

The signaling molecule auxin is pivotal in coordinating many growth and development processes in plants mainly through the modulation of gene expression. The transcriptional response to auxin is mediated by the family of auxin response factors (ARF). Monomers of this family recognize a DNA motif (TGTC[TC]/[GG]) called the auxin-response element (AuxRE). ARFs can homodimerize through their DNA binding domains (DBD) thereby enabling cooperative binding for a bipartite inverted AuxRE (IR7). In addition to the DBD, most ARFs contain a C-terminal Phox and Bem1p (PB1) domain both capable of homotypic interactions, and mediating interactions with Aux/IAA repressors. Given the dual role of the PB1 domain, and the ability of both DBD and PB1 domain to mediate dimerization, a key question is how each of these domains contributes to conferring DNA-binding specificity and affinity. So far, ARF-ARF and ARF-DNA interactions have mostly been approached using qualitative methods that do not provide a quantitative and dynamic view on the binding equilibria. Here, we utilize a DNA binding assay based on single-molecule Förster resonance energy transfer (smFRET) to study the affinity and kinetics of the interaction of severalArabidopsis thalianaARFs with an IR7 AuxRE. We show that both DBD and PB1 domains of AtARF2 contribute toward DNA binding, and we identify ARF dimer stability as a key parameter in defining affinity and kinetics seen for the DBDs of different AtARFs. Lastly, we derived an analytical solution for a four-state cyclic model that explains both the kinetics and the affinity of the interaction between AtARF2 and IR7. Our work demonstrates that the affinity of ARFs towards composite DNA response elements can be tuned by small changes of their dimerization equilibrium suggesting that this effect has major implications for ARF-mediated transcriptional activity.

The stratum corneum's lipid matrix is a critical for the skin's barrier function and is primarily composed of ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs). The lipids form a long periodicity phase (LPP), a unique trilayer unit cell structure. An enzyme driven pathway is implemented to synthesize these key lipids. If these enzymes are down- or upregulated as in inflammatory diseases, the final lipid composition is affected often altering the barrier function. In this study, we mimicked down regulation of enzymes involved in the synthesis of the sphingosine and CER amide bond. In a LPP lipid model, we substituted CER N-(tetracosanoyl)- sphingosine (CER NS) with either i) FFA C24 and free sphingosine, to simulate the loss of the CER amide bond, or ii) with FFA C24 and C18 to simulate the loss of the sphingosine headgroup. Our study shows the lipids in the LPP would not phase separate until at least 25% of the CER NS is substituted keeping the lateral packing and conformational ordering unaltered. Neutron diffraction studies showed that free sphingosine chains localized at the outer layers of the unit cell, while the remaining CER NS head group was concentrated in the inner headgroup layers. However, when FFA C18 was inserted, CER NS was dispersed throughout the LPP, resulting in an even distribution between the inner and outer water layers. The presented results highlight the importance of the CER NS headgroup structure and its interaction in combination with the carbon chain invariability for optimal lipid arrangement.

The addition of compounds to scavenge the radical species produced during biological small-angle X-ray scattering (BioSAXS) experiments is a common strategy to reduce the effects of radiation damage and produce better quality data. As almost half of the experiments leading to structures deposited in the SASBDB database used scavengers, finding potent scavengers would be advantageous for many experiments. Here, four compounds, three nucleosides and one nitrogenous base, are presented which can act as very effective radical-scavenging additives and increase the critical dose by up to 20 times without altering the stability or reducing the contrast of the tested protein solutions. The efficacy of these scavengers is higher than those commonly used in the field to date, as verified for lysozyme solutions at various concentrations from 7.0 to 0.5 mg ml−1. The compounds are also very efficient at mitigating radiation damage to four proteins with molecular weights ranging from 7 to 240 kDa and pH values from 3 to 8, with the extreme case being catalase at 6.7 mg ml−1, with a scavenging factor exceeding 100. These scavengers can therefore be instrumental in expanding BioSAXS to low-molecular-weight and low-concentration protein samples that were previously inaccessible owing to poor data quality. It is also demonstrated that an increase in the critical dose in standard BioSAXS experiments leads to an increment in the retrieved information, in particular at higher angles, and thus to higher resolution of the model.

15 p.-4 fig.-3 tab., Quorum sensing allows bacterial cells to communicate through the release of soluble signaling molecules into the surrounding medium. It plays a pivotal role in controlling bacterial conjugation in Gram-positive cells, a process that has tremendous impact on health. Intracellular regulatory proteins of the RRNPP family are common targets of these signaling molecules. The RRNPP family of gene regulators bind signaling molecules at their C-terminal domain (CTD), but have highly divergent functionalities at their N-terminal effector domains (NTD). This divergence is also reflected in the functional states of the proteins, and is highly interesting from an evolutionary perspective. RappLS20 is an RRNPP encoded on the Bacillus subtilis plasmid pLS20. It relieves the gene repression effectuated by RcopLS20 in the absence of the mature pLS20 signaling peptide Phr*pLS20. We report here an in-depth structural study of apo and Phr*pLS20-bound states of RappLS20 at various levels of atomic detail. We show that apo-RappLS20 is dimeric and that Phr*pLS20-bound Rap forms NTD-mediated tetramers. In addition, we show that RappLS20 binds RcopLS20 directly in the absence of Phr*pLS20 and that addition of Phr*pLS20 releases RcopLS20 from RappLS20. This allows RcopLS20 to bind the promotor region of crucial conjugation genes blocking their expression., Ministry of Economy and Competitiveness of the Spanish Government [BFU2016-75471-C2-1-P (AEI/FEDER,EU) to C.A., BIO2013-41489-P (AEI/FEDER, EU) to W.M. which also funded AM-A, BIO2016-77883-C2-2-P (AEI/FEDER, EU) to R.B., BIO2016-77883-C2-2-P(AEI/FEDER, EU) also supported N.B.]; ‘Fundación Ramón Areces’ and ‘Banco de Santander’ to the Centro de Biología Molecular ‘Severo Ochoa’.Funding for open access charge: Ministry of Economy and Competitiveness of the Spanish Government [BIO2016-77883-C2-1-P and BIO2016-77883-C2-2-P].

We study the particle size distribution and phase changes of the anatase TiO2 nanopowder samples when they are subject to the plasma treatments of three different kinds of gases as nitrogen (N2), oxygen (O2), and argon (Ar). The plasma gas pressures vary as 0.1, 0.3, and 0.6 Torr. We demonstrate that the plasma treatments have an effect neither on the phase structure nor on the mean nanocrystalline size. The phase and size invariances of the samples are attributed to their nanoscale thermodynamic aspects. We find out that elevating the gas pressure in some cases creates fine-size amorphous nanoparticles with a narrow distribution. Our findings authenticate that plasma treatment affects the amorphous phase with etching particles down to a mean value of ∼3 nm. The small-angle X-ray scattering (SAXS) technique was utilized to obtain the size distribution of the nanoparticles, and the wide-angle X-ray scattering (WAXS) technique was used to probe the phase and size changes of the crystalline structure.

Photoelectrochemical cells that utilize water as a source of electrons are one of the most attractive solutions for the replacement of fossil fuels by clean and sustainable solar fuels. To achieve this, heterogeneous water oxidation catalysis needs to be mastered and properly understood. The search continues for a catalyst that is stable at the surface of electro(photo)anodes and can efficiently perform this reaction at the desired neutral pH. Here, we show how oligomeric Ru complexes can be anchored on the surfaces of graphitic materials through CH–π interactions between the auxiliary ligands bonded to Ru and the hexagonal rings of the graphitic surfaces, providing control of their molecular coverage. These hybrid molecular materials behave as molecular electroanodes that catalyse water oxidation to dioxygen at pH 7 with high current densities. This strategy for the anchoring of molecular catalysts on graphitic surfaces can potentially be extended to other transition metals and other catalytic reactions. Efficient and stable water oxidation catalysts are important if photoelectrochemical cells are to be used to provide clean and sustainable solar fuels. A water oxidation catalyst that operates at neutral pH has now been developed that features ruthenium coordination oligomers anchored onto the surfaces of graphitic materials through CH–π interactions.

Anti

Bacterial conjugation is an important route for horizontal gene transfer. The initial step in this process involves a macromolecular protein-DNA complex called the relaxosome, which in plasmids consists of the origin of transfer (oriT) and several proteins that prepare the transfer. The relaxosome protein named relaxase introduces a nick in one of the strands of the oriT to initiate the process. Additional relaxosome proteins can exist. Recently, several relaxosome proteins encoded on the Bacillus subtilis plasmid pLS20 were identified, including the relaxase, named RelpLS20, and two auxiliary DNA-binding factors, named Aux1pLS20 and Aux2pLS20. Here, we extend this characterization in order to define their function. We present the low-resolution SAXS envelope of the Aux1pLS20 and the atomic X-ray structure of the C-terminal domain of Aux2pLS20. We also study the interactions between the auxiliary proteins and the full-length RelpLS20, as well as its separate domains. The results show that the quaternary structure of the auxiliary protein Aux1pLS20 involves a tetramer, as previously determined. The crystal structure of the C-terminal domain of Aux2pLS20 shows that it forms a tetramer and suggests that it is an analog of TraMpF of plasmid F. This is the first evidence of the existence of a TraMpF analog in gram positive conjugative systems, although, unlike other TraMpF analogs, Aux2pLS20 does not interact with the relaxase. Aux1pLS20 interacts with the C-terminal domain, but not the N-terminal domain, of the relaxase RelpLS20. Thus, the pLS20 relaxosome exhibits some unique features despite the apparent similarity to some well-studied G- conjugation systems.

12 pags., 8 figs., We have investigated the crystallization of the thermoplastic polymer Poly(vinylidene Fluoride) (PVDF) during “operando” 3D printing Fused Filament Fabrication (FFF). The performance of the 3D printing set-up and the corresponding methodology for performing simultaneous SAXS/WAXS with synchrotron radiation have been discussed. Simultaneous SAXS and WAXS experiments were performed across the printed line with a resolution of 50 μm. The experiments indicate that crystallization is faster at the polymer-air interface than in other points within the printed line. The final crystallinity varies with position being lower at both the interfaces, i.e. polymer-air (13%) and welding zone (11%), while being higher (18%) in the middle part of the printed line. Orientation of the crystalline lamellae is higher at both interfaces, suggesting higher shear rate than in the bulk of the printed line where elongational flow is dominant. The final crystallinity levels in the different locations of the printed line are relatively low, as compared with reported values in melt pressed samples (≈30%), due to the extremely fast crystallization kinetics involved in the solidification of PVDF 3D printed lines. Therefore, it is expected that 3D printed PVDF pieces will exhibit significant structural modifications due to the potential crystallization that eventually will proceed during storage because the glass transition of PVDF is well below room temperature., This work has been supported by Agencia Estatal de Investigación(AEI) through projects: PID2019 - 107514 GB - I00/AEI/10.13039/501100011033 and PID2019 - 106125 GB -I00/AEI/10.13039/501100011033. The experiments with synchrotron radiation were performed at NCD-SWEET beamline at ALBA Synchrotron with the collaboration of ALBA staff (Proposal 2020024176).

This study reports on the synthesis of novel bienzyme polymer-assisted nanoflower complexes (PANF), their morphological and structural characterization, and their effectiveness as cascade biocatalysts. First, highly porous polyamide 6 microparticles (PA6 MP) are synthesized by activated anionic polymerization in solution. Second, the PA6 MP are used as carriers for hybrid bienzyme assemblies comprising glucose oxidase (GOx) and horseradish peroxidase (HRP). Thus, four PANF complexes with different co-localization and compartmentalization of the two enzymes are prepared. In samples NF GH/PA and NF GH@PA, both enzymes are localized within the same hybrid flowerlike organic-inorganic nanostructures (NF), the difference being in the way the PA6 MP are assembled with NF. In samples NF G/PAiH and NF G@PAiH, only GOx is located in the NF, while HRP is preliminary immobilized on PA6 MP. The morphology and the structure of the four PANF complexes have been studied by microscopy, spectroscopy, and synchrotron X-ray techniques. The catalytic activity of the four PANF was assessed by a two-step cascade reaction of glucose oxidation. The PANF complexes are up to 2–3 times more active than the free GOx/HRP dyad. They also display enhanced kinetic parameters, superior thermal stability in the 40–60 °C range, optimum performance at pH 4–6, and excellent storage stability. All PANF complexes are active for up to 6 consecutive operational cycles., The authors gratefully acknowledge the financial support of Fundação para a Ciência e Tecnologia (FCT), project UID/CTM/50025/2019. Special thank is due to the ALBA synchrotron governance for financing our WAXS/SAXS experiments at NCD SWEET beamline in the framework of the project ID 2018/022726. N. Dencheva is also grateful for the financial support of FCT in the frames of the personal program contract CTTI-51/18-IPC. J. Braz acknowledges the support of the FCT UI/BD/150854/2021 Ph.D. grant.

In situ research of materials under moderate pressures (hundreds of bar) is essential in many scientific fields. These range from gas sorption to chemical and biological processes. One industrially important discipline is the hydration of oil well cements. Existing capillary cells in this pressure range are static as they are easy to design and operate. This is convenient for the study of single-phase materials; however, powder diffraction quantitative analyses for multiphase systems cannot be performed accurately as a good powder average cannot be attained. Here, the design, construction and commissioning of a cost-effective spinning capillary cell for in situ powder X-ray diffraction is reported, for pressures currently up to 200 bar. The design addresses the importance of reducing the stress on the capillary by mechanically synchronizing the applied rotation power and alignment on both sides of the capillary while allowing the displacement of the supports needed to accommodate different capillaries sizes and to insert the sample within the tube. This cell can be utilized for multiple purposes allowing the introduction of gas or liquid from both ends of the capillary. The commissioning is reported for the hydration of a commercial oil well cement at 150 bar and 150°C. The quality of the resulting powder diffraction data has allowed in situ Rietveld quantitative phase analyses for a hydrating cement containing seven crystalline phases.

Anti-EFG1 2-OMethylRNA is an antisense oligonucleotide (ASO) that has the ability to recognize and block the EFG1 gene and to control Candida albicans filamentation. However, it is important to protect the anti-EFG1 2-OMethylRNA ASO from the environmental human body conditions and to ensure that they will be delivered to their site of action, and polyplex microparticles (MPs) represent a class of vehicles to ASO cargo with these functionalities. Thus, the goal of this work was to develop polyplexes based on porous poly(-butyrolactam) (PA4) or poly(-caprolactam) (PA6) MPs for the anti-EFG1 2-OMethylRNA ASO cargo and delivery. Two types of polyplexes were prepared with payloads of anti-EFG1 2-OMethylRNA molecules, either entrapped or immobilized on prefabricated polyamide MPs. Our data confirm that PA4 and PA6 polyplex MPs can be feasible carriers for anti-EFG1 2-OMethylRNA ASO molecules, using either the entrapment or immobilization strategies, whereby the released ASO maintains its activity against C. albicans cells., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of the UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020Programa Operacional Regional do Norte and Daniela Eira Araujo [SFRH/BD/121417/2016] Ph.D. grant. The authors also acknowledge the project funding by the “02/SAICT/2017 Projetos de Investigação Científica e Desenvolvimento Tecnológico (IC&DT) POCI-01-0145-FEDER-028893”. N.V.D. and Z.Z.D. are also grateful to the Executive Commission of ALBA synchrotron for the support under the project no. 2018022726., info:eu-repo/semantics/publishedVersion

The skin’s barrier ability is an essential function for terrestrial survival, which is controlled by intercellular lipids within the stratum corneum (SC) layer. In this barrier, free fatty acids (FFAs) are an important lipid class. As seen in inflammatory skin diseases, when the lipid chain length is reduced, a reduction in the barrier’s performance is observed. In this study, we have investigated the contributing effects of various FFA chain lengths on the lamellar phase, lateral packing. The repeat distance of the lamellar phase increased with FFA chain length (C20–C28), while shorter FFAs (C16 to C18) had the opposite behaviour. While the lateral packing was affected, the orthorhombic to hexagonal to fluid phase transitions were not affected by the FFA chain length. Porcine SC lipid composition mimicking model was then used to investigate the proportional effect of shorter FFA C16, up to 50% content of the total FFA mixture. At this level, no difference in the overall lamellar phases and lateral packing was observed, while a significant increase in the water permeability was detected. Our results demonstrate a FFA C16 threshold that must be exceeded before the structure and barrier function of the long periodicity phase (LPP) is affected. These results are important to understand the lipid behaviour in this unique LPP structure as well as for the understanding, treatment, and development of inflammatory skin conditions.

Bacterial conjugation is an important route for horizontal gene transfer. The initial step in this process involves a macromolecular protein-DNA complex called the relaxosome, which in plasmids consists of the origin of transfer (

Thermoplastic polyurethanes (TPU) with varying hard segment contents (HSC) are monitored during melting and solidifying (20 K/min , Tmax = 220 ° C) by small-angle and wide-angle X-ray scattering (WAXS and SAXS). Hard segments: MDI/BD. Soft segments: PTHF1000. The neat materials are injection-molded, having small amorphous hard domains (chord length d⎯⎯h ∼ 35% show sharp Bragg peaks and larger hard domains ( d⎯⎯h > 7 nm ). When heated, small domains melt, but crystallization in the remaining large domains is not detected. Upon cooling, large agglomerates segregate first, which crystallize immediately. Segregation starts for HSC = 42% at 160 °C and for HSC = 75% at 210 °C. When HSC ≤ 30%, the morphologies before and after are similar, but afterward, many hard blocks are dissolved in the soft phase at the expense of the hard domain fraction. In heating and cooling the melts, multiple homogenization and segregation processes are observed, which are explained by the agglomeration of hard blocks of different lengths in the colloidal fluid.

We study the particle size distribution and phase changes of the anatase TiO2 nanopowder samples when they are subject to the plasma treatments of three different kinds of gases as nitrogen (N2), oxygen (O2), and argon (Ar). The plasma gas pressures vary as 0.1, 0.3, and 0.6 Torr. We demonstrate that the plasma treatments have an effect neither on the phase structure nor on the mean nanocrystalline size. The phase and size invariances of the samples are attributed to their nanoscale thermodynamic aspects. We find out that elevating the gas pressure in some cases creates fine-size amorphous nanoparticles with a narrow distribution. Our findings authenticate that plasma treatment affects the amorphous phase with etching particles down to a mean value of ∼3 nm. The small-angle X-ray scattering (SAXS) technique was utilized to obtain the size distribution of the nanoparticles, and the wide-angle X-ray scattering (WAXS) technique was used to probe the phase and size changes of the crystalline structure.

Structural and morphological interplay between hard and soft phases determine the bulk properties of thermoplastic polyurethanes. Commonly employed techniques rely on different physical or chemical phenomena for characterizing the organization of domains, but detailed structural information can be difficult to derive. Here, total scattering pair distribution function (PDF) analysis is used to determine atomic-scale insights into the connectivity and molecular ordering and compared to the domain size and morphological characteristics measured by AFM, TEM, SAXS, WAXS, and solid-state NMR 1H–1H spin-diffusion. In particular, density distribution functions are highlighted as a means to bridging the gap from the domain morphology to intradomain structural ordering. High real-space resolution PDFs are shown to provide a sensitive fingerprint for indexing aromatic, aliphatic, and polymerization-induced bonding characteristics, as well as the hard phase structure, and indicate that hard phases coexist in both ordered and disordered states.

This study reports a new strategy for enzyme immobilization based on passive immobilization in neat and magnetically responsive polyamide 4 (PA4) highly porous particles. The microsized particulate supports were synthesized by low-temperature activated anionic ring-opening polymerization. The enzyme of choice was laccase from Trametes versicolor and was immobilized by either adsorption on prefabricated PA4 microparticles (PA4@iL) or by physical in situ entrapment during the PA4 synthesis (PA4@eL). The surface topography of all PA4 particulate supports and laccase conjugates, as well as their chemical and physical structure, were studied by microscopic, spectral, thermal, and synchrotron WAXS/SAXS methods. The laccase content and activity in each conjugate were determined by complementary spectral and enzyme activity measurements. PA4@eL samples displayed &gt, 93% enzyme retention after five incubation cycles in an aqueous medium, and the PA4@iL series retained ca. 60% of the laccase. The newly synthesized PA4-laccase complexes were successfully used in dyestuff decolorization aiming at potential applications in effluent remediation. All of them displayed excellent decolorization of positively charged dyestuffs reaching ~100% in 15 min. With negative dyes after 24 h the decolorization reached 55% for PA4@iL and 85% for PA4@eL. A second consecutive decolorization test revealed only a 5&ndash, 10% decrease in effectiveness indicating the reusability potential of the laccase-PA4 conjugates.

Melting and solidification of thermoplastic polyurethanes (TPU) are monitored by X-ray scattering. The colloidal character of the typical material becomes unstable as soon as the hard domains (HD) are molten. Then the scattered intensity fluctuates considerably and “lost” photons are found at small scattering angles (Tyndall effect). In this temperature regime the material is a fluid. Competing homogenization and segregation processes appear to take place, which modulate the materials colloidal character. Monitored by SAXS and WAXS, TPU elastomers are melted and re-solidified (heating rate: 20 K/min, temperatures T m a x : 220 °C, 190 °C). Strong fluctuations in scattering intensity due to varying Tyndall effect are observed in most of our experiments. Here the typical effects are demonstrated on a polyether-based TPU with a hard segment content of 30%, whose colloidal melt is stabilized by adding 0.5% graphite. In the hot quiescent melt the morphology is characterized by density undulations. HD arrangement does not grow late. Graphite stabilizes the colloidal melt. It increases both the HD melting-temperature (from 185 °C to 210 °C) and the HD formation temperature (from 90 °C to 165 °C).

Macromolecular crystallography (MX) and small-angle X-ray scattering (SAXS) studies on proteins at synchrotron light sources are commonly limited by the structural damage produced by the intense X-ray beam. Several effects, such as aggregation in protein solutions and global and site-specific damage in crystals, reduce the data quality or even introduce artefacts that can result in a biologically misguiding structure. One strategy to reduce these negative effects is the inclusion of an additive in the buffer solution to act as a free radical scavenger. Here the properties of uridine as a scavenger for both SAXS and MX experiments on lysozyme at room temperature are examined. In MX experiments, upon addition of uridine at 1 M, the critical dose D 1/2 is increased by a factor of ∼1.7, a value similar to that obtained in the presence of the most commonly used scavengers such as ascorbate and sodium nitrate. Other figures of merit to assess radiation damage show a similar trend. In SAXS experiments, the scavenging effect of 40 mM uridine is similar to that of 5% v/v glycerol, and greater than 2 mM DTT and 1 mM ascorbic acid. In all cases, the protective effect of uridine is proportional to its concentration.

Microstructure formation in individual layers during fused filament fabrication (FFF) of a Π-shaped multilayer single-walled polymer sample was studied by simultaneous measurement of small- and wide-angle X-ray scattering (SAXS and WAXS, respectively) methods employing synchrotron radiation. We investigated individual layers and the welding zone between individual layers. As a model material, we used isotactic polypropylene (iPP), which is a commodity semicrystalline polymer and has a strong potential as a feedstock material for additive manufacturing. The layers were deposited by an FFF three-dimensional (3D) printer that was custom-built to fit into the synchrotron beamline. WAXS data were utilized to determine the temperature of the irradiated volume. The polymer microstructure was characterized in terms of crystallinity and long-spacing. Avrami analysis indicates that the crystallization behavior of iPP in thin layers is rather similar to that observed in quiescent crystallization of bulk iPP, suggesting similar nucleation and growth mechanisms. Our results revealed a variation of crystallinity across the individual layers, reflecting the influence of interfaces (free surface and welding zone) on the final crystallinity of the thin layer (thickness h = 0.02 cm): the polymer is more crystalline in the bulk of the layer and less crystalline in the vicinity of the interfaces. This effect can be advantageous to facilitate the welding between the layers, i.e., to improve the overall mechanical performance of the 3D-printed object because welding between layers is expected to occur by polymer chain interdiffusion. Along the thin layer we observed a higher crystallinity near the corners attributed to a deceleration of the print head.

The synthesis of three gold(I) tripodal complexes derived from tripropargylamine and containing the water soluble phosphines PTA (1, 3,5-triaza-7-phosphaadamantane), DAPTA (3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane) and TPPTS (triphenylphosfine-3,3',3''-trisulfonic acid trisodium salt) is here described. The three complexes are observed to give rise to the formation of supramolecular aggregates in water and very long fibers. This property has been analyzed by means of 1H-NMR spectroscopy at different concentrations and SAXS. The results point out the important role of the phosphine moieties as the main enthalpic or entropic contribution in the resulting Gibbs energy of aggregates formation. The tripodal structure of the three complexes together with the presence of gold(I) atoms make them ideal candidates to interact with hydrophobic molecules also in water. For this, the interaction with pyrene in this solvent has been evaluated with successful results in all three complexes. The highest association constant corresponds to 2 as the host. DFT studies indicates the location of pyrene in the tripodal cavity as the most stable conformation. The interaction with pyrene has been additionally studied within cholate hydrogel matrixes pointing out the stability of the resulting host:guest adducts in the different medium.

Herewith we report the first attempt towards non-covalent immobilization of Trametes versicolor laccase on neat and magnetically responsive highly porous polyamide 6 (PA6) microparticles and their application for catechol oxidation. Four polyamide supports, namely neat PA6 and such carrying Fe, phosphate-coated Fe and Fe3O4 cores were synthesized in suspension by activated anionic ring-opening polymerization (AAROP) of ε-caprolactam (ECL). Enzyme adsorption efficiency up to 92% was achieved in the immobilization process. All empty supports and PA6 laccase complexes were characterized by spectral and synchrotron WAXS/SAXS analyses. The activity of the immobilized laccase was evaluated using 2,2’-Azino-bis-(3- ethylbenzothiazoline-6-sulfonic acid (ABTS) and compared to the native enzyme. The PA6 laccase conjugates displayed up to 105% relative activity at room temperature, pH 4, 40 °C and 20 mM ionic strength (citrate buffer). The kinetic parameters of the ABTS oxidation were also determined. The reusability of the immobilized laccase-conjugates was proven for five consecutive oxidation cycles of catechol., The authors gratefully acknowledge the financial support of the project TSSiPRO NORTE01-0145-FEDER-000015, supported by the regional operation program NORTE2020, under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund, as well as the support by National Funds through Fundação para a Ciência e Tecnologia (FCT), project UID/CTM/50025/2019. D.G. and I.G. wish to thank the Research Foundation of the State of New York—Networks of Excellence and the McIntyre-Stennis program of the US Department of Agriculture for partial funding. N.D. is also grateful for the personal program-contract CTTI-51/18-IPC.

This work presents the preparation, optimization, and testing of an enzymebased optical biosensor for catechol determination. The sensing area is attached to a glass support and contains: anionic polyamide 6 (PA6) porous microparticles supporting laccase from Trametes Versicolor, embedded in a Pebax® MH1657 polymer binder that contains the optical indicator dye 3-methyl-2-benzothiazolinone hydrazone (MBTH), responsible for the optical transduction. The catechol analyte, after its enzymatic oxidation, forms o-benzoquinone that can be detected by oxidative coupling with MBTH giving rise to a colored product. The latter can be quantified easuring the UV/VIS absorbance at 500 nm. The PA6 microparticles performed as useful laccase carriers reaching high immobilization yields of up to 99.8% and preserving the enzyme catalytic activity. This permitted the reparation of a new biosensor presenting a detection limit of 11 μM and responding linearly to up to 118 μM of catechol. Biosensor applicability was tested in spiked natural water samples from river and spring. The recovery rates observed were in the range of 97–108% that proves the good accuracy of the proposed biosensor., All authors gratefully acknowledge the financial support of the project TSSiPRO NORTE-01-0145-FEDER-000015, supported by the regional operation program NORTE2020, under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund, as well as the support by National Funds through Fundação para a Ciência e Tecnologia (FCT), project UID/CTM/50025/2019. Special thank is due to the ALBA synchrotron governance for financing our WAXS/SAXS experiments at NCD SWEET beamline in the framework of the approved pro posal ID 2018/022726. N. Dencheva is also grateful for the financial support of FCT in the frames of the strategic project UID/CTM/50025/2013 and the personal program contract CTTI-51/18-IPC. The authors gratefully acknowledge the support of Dr. Amélie Noel (Arkema, France) for providing a free sample of Pebax® MH 1657.

The reaction of the gold polymers containing bipyridyl and terpyridyl units, [Au(C≡CC15H10N3)]n and [Au(C≡CC10H7N2)]n, with the water-soluble phosphines 1,3,5-triaza-7-phosphatricyclo[3.3.1.13.7]decane and 3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane gives rise to the formation of four gold(I) alkynyl complexes that self-assemble in water (H2O) and dimethyl sulfoxide (DMSO), through different intermolecular interactions, with an impact on the observed luminescence displayed by the supramolecular assemblies. A detailed analysis carried out by NMR studies performed in different DMSO/deuterated H2O mixtures indicates the presence of two different assembly modes in the aggregates: (i) chain assemblies, which are based mainly on aurophilic interactions, and (ii) stacked assemblies, which are based on Au···π and π···π interactions. These different supramolecular environments can also be detected by their intrinsic optical properties (differences in absorption and emission spectra) and are predicted by the changes in the relative binding energy from density functional theory calculations carried out in DMSO and H2O. Small-angle X-ray scattering (SAXS) experiments performed in the same mixture of solvents are in agreement with the formation of aggregates in all cases. The aromatic units chosen, bipyridine and terpyridine, allow the use of external stimuli to reversibly change the aggregation state of the supramolecular assemblies. Interaction with the Zn2+ cation is observed to disassemble the aggregates, while encapsulating agents competing for Zn2+ complexation revert the process to the aggregation stage, as verified by SAXS and NMR. The adaptive nature of the supramolecular assemblies to the metal-ion content is accompanied by significant changes in the absorption and emission spectra, signaling the aggregation state and also the content on Zn2+. © 2017 American Chemical Society., The authors are grateful to the Ministry of Economy, Industry and Competitiveness of Spain (AEI/FEDER, UE Projects CTQ2016-76120-P, CTQ2015-65040-P, and CTQ2015-70117-R). This work was also supported by the Associated Laboratory for Sustainable Chemistry, Clean Processes and Technologies, LAQV, which is financed by national funds from FCT/MEC (UID/QUI/50006/2013) and cofinanced by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007265). The IQAC NMR Facility 500 MHz spectrometer and cryoprobe were funded by the CSIC13-4E-2076 MINECO-FEDER grant. SAXS experiments were performed at the NCD-BL11 beamline of the ALBA Synchrotron Light Facility in collaboration with the ALBA staff. A.J.M. thanks the FCT for a postdoctoral grant (SFRH/ BPD/69210/2010). This research was supported by a Marie Curie Intra European Fellowship within the 7th European Community Framework Programme (to R.G.). We also thank L. Gimeńez and I. Brea for their help on the synthesis of the ligands.

Coarse-grained simulation models are developed to study both template-bound and free porphyrin nanoring systems. Key interactions are modeled with relatively simple (and physically motivated) energy functions which allow for relatively facile transfer both between different ring sizes and between the template-bound and free nanoring systems. The effects of varying the model parameters on the respective radii of gyration are determined. The effects of including different templates on the ring structure are investigated both in terms of the detailed geometry of the template and the interaction strength between the template and the metal centers in the nanorings. The role of the template-nanoring interaction strength in controlling potential "caterpillar track" rotational motion is discussed. The relationship of the model to experimental small-angle X-ray, exchange spectroscopy, and electron spin resonance results is discussed.

Vernier templating exploits a mismatch between the number of binding sites in a template and a reactant to direct the formation of a product that is large enough to bind several template units. Here, we present a detailed study of the Vernier-templated synthesis of a 12-porphyrin nanoring. NMR and small-angle X-ray scattering (SAXS) analyses show that Vernier complexes are formed as intermediates in the cyclo-oligomerization reaction. UV/Vis/NIR titrations show that the three-component assembly of the 12-porphyrin nanoring figure-of-eight template complex displays high allosteric cooperativity and chelate cooperativity. This nanoring–template 1:2 complex is among the largest synthetic molecules to have been characterized by single-crystal analysis. It crystallizes as a racemate, with an angle of 27° between the planes of the two template units. The crystal structure reveals many unexpected intramolecular C[BOND]H⋅⋅⋅N contacts involving the tert-butyl side chains. Scanning tunneling microscopy (STM) experiments show that molecules of the 12-porphyrin template complex can remain intact on the gold surface, although the majority of the material unfolds into the free nanoring during electrospray deposition.

Measurements of a well-characterised standard sample can verify the performance of an instrument. Typically, small-angle neutron scattering instruments are used to investigate a wide range of samples and may often be used in a number of configurations. Appropriate standard samples are useful to test different aspects of the performance of hardware as well as that of the data reduction and analysis software. Measurements on a number of instruments with different intrinsic characteristics and designs in a round robin can not only better characterise the performance for a wider range of conditions but also, perhaps more importantly, reveal the limits of the current state of the art of small-angle scattering. The exercise, followed by detailed analysis, tests the limits of current understanding as well as uncovers often forgotten assumptions, simplifications and approximations that underpin the current practice of the technique. This paper describes measurements of polystyrene latex, radius 72 nm with a number of instruments. Scattering from monodisperse, uniform spherical particles is simple to calculate and displays sharp minima. Such data test the calibrations of intensity, wavelength and resolution as well as the detector response. Smoothing due to resolution, multiple scattering and polydispersity has been determined. Sources of uncertainty are often related to systematic deviations and calibrations rather than random counting errors. The study has prompted development of software to treat modest multiple scattering and to better model the instrument resolution. These measurements also allow checks of data reduction algorithms and have identified how they can be improved. The reproducibility and the reliability of instruments and the accuracy of parameters derived from the data are described., Comment: Journal of Applied Crystallography - accepted for publication

Chemists use template-directed synthesis to position molecular components so that they can be covalently linked into complex molecules that are not readily accessible by classical synthetic methods. But as larger structures are targeted, the synthesis of the templates themselves becomes challenging. O'Sullivan et al. now show that 'molecular Verniers', based on the principle of moire pattern formation, can solve this problem. Using a Vernier template with six binding sites and molecular building blocks with four porphyrins acting as binding sites, the authors create a 12-porphyrin nano-ring with a diameter of 4.7 nanometres. The ease and efficiency of this synthesis establishes Vernier templating as a powerful new strategy for producing large monodisperse macromolecules. Templates are widely used to arrange molecular components so they can be covalently linked into complex molecules that are not readily accessible by classical synthetic methods. But, as larger structures are targeted, the synthesis of the templates themselves becomes challenging. It is now shown that 'molecular Verniers' can solve this problem: using a template with six binding sites and molecular building blocks with four porphyrins acting as binding sites, a 12-porphyrin nano-ring with a diameter of 4.7 nm is created. The ease and efficiency of this synthesis establishes Vernier templating as a powerful new strategy for producing large monodisperse macromolecules. Templates are widely used to arrange molecular components so they can be covalently linked into complex molecules that are not readily accessible by classical synthetic methods1,2,3,4,5,6,7. Nature uses sophisticated templates such as the ribosome, whereas chemists use simple ions or small molecules. But as we tackle the synthesis of larger targets, we require larger templates—which themselves become synthetically challenging. Here we show that Vernier complexes can solve this problem: if the number of binding sites on the template, nT, is not a multiple of the number of binding sites on the molecular building blocks, nB, then small templates can direct the assembly of relatively large Vernier complexes where the number of binding sites in the product, nP, is the lowest common multiple of nB and nT (refs 8, 9). We illustrate the value of this concept for the covalent synthesis of challenging targets by using a simple six-site template to direct the synthesis of a 12-porphyrin nano-ring with a diameter of 4.7 nm, thus establishing Vernier templating as a powerful new strategy for the synthesis of large monodisperse macromolecules.

Linear π-conjugated oligomers have been widely investigated, but the behavior of the corresponding cyclic oligomers is poorly understood, despite the recent synthesis of π-conjugated macrocycles such as [n]cycloparaphenylenes and cyclo[n]thiophenes. Here we present an efficient template-directed synthesis of a π-conjugated butadiyne-linked cyclic porphyrin hexamer directly from the monomer. Small-angle X-ray scattering data show that this nanoring is shape-persistent in solution, even without its template, whereas the linear porphyrin hexamer is relatively flexible. The crystal structure of the nanoring-template complex shows that most of the strain is localized in the acetylenes; the porphyrin units are slightly curved, but the zinc coordination sphere is undistorted. The electrochemistry, absorption, and fluorescence spectra indicate that the HOMO-LUMO gap of the nanoring is less than that of the linear hexamer and less than that of the corresponding polymer. The nanoring exhibits six one-electron reductions and six one-electron oxidations, most of which are well resolved. Ultrafast fluorescence anisotropy measurements show that absorption of light generates an excited state that is delocalized over the whole π-system within a time of less than 0.5 ps. The fluorescence spectrum is amazingly structured and red-shifted. A similar, but less dramatic, red-shift has been reported in the fluorescence spectra of cycloparaphenylenes and was attributed to a high exciton binding energy; however the exciton binding energy of the porphyrin nanoring is similar to those of linear oligomers. Quantum-chemical excited state calculations show that the fluorescence spectrum of the nanoring can be fully explained in terms of vibronic Herzberg-Teller (HT) intensity borrowing.

International audience; The J aggregates of 4-sulfonatophenyl meso-substituted porphyrins are non-covalent polymers obtained by self-assembly that form nanoparticles of different morphologies. In the case of high aspect-ratio nanoparticles (bilayered ribbons and monolayered nanotubes), shear hydrodynamic forces may modify their shape and size, as observed by peak force microscopy, transmission electron microscopy of frozen solutions, small-angle X-ray scattering measurements in a disk-plate rotational cell, and cone plate rotational viscometry. These nanoparticles either show elastic or plastic behaviour: there is plasticity in the ribbons obtained upon nanotube collapse on solid/air interfaces and in viscous concentrated nanotube solutions, whereas elasticity occurs in the case of dilute nanotube solutions. Sonication and strong shear hydrodynamic forces lead to the breaking of the monolayered nanotubes into small particles, which then associate into large colloidal particles.

The role of Decorin in organising the extracellular matrix was examined in normal human corneas and in corneas from patients with Congenital Stromal Corneal Dystrophy (CSCD). In CSCD, corneal clouding occurs due to a truncating mutation (c.967delT) in the decorin (DCN) gene. Normal human Decorin protein and the truncated one were reconstructed in silico using homology modelling techniques to explore structural changes in the diseased protein. Corneal CSCD specimens were also examined using 3-D electron tomography and Small Angle X-ray diffraction (SAXS), to image the collagen-proteoglycan arrangement and to quantify fibrillar diameters, respectively. Homology modelling showed that truncated Decorin had a different spatial geometry to the normal one, with the truncation removing a major part of the site that interacts with collagen, compromising its ability to bind effectively. Electron tomography showed regions of abnormal stroma, where collagen fibrils came together to form thicker fibrillar structures, showing that Decorin plays a key role in the maintenance of the order in the normal corneal extracellular matrix. Average diameter of individual fibrils throughout the thickness of the cornea however remained normal.

The CCN (cyr61, ctgf, nov) proteins (CCN1–6) are an important family of matricellular regulatory factors involved in internal and external cell signaling. They are central to essential biological processes such as adhesion, proliferation, angiogenesis, tumorigenesis, wound healing, and modulation of the extracellular matrix. They possess a highly conserved modular structure with four distinct modules that interact with a wide range of regulatory proteins and ligands. However, at the structural level, little is known although their biological function(s) seems to require cooperation between individual modules. Here we present for the first time structural determinants of two of the CCN family members, CCN3 and CCN5 (expressed in Escherichia coli), using small angle x-ray scattering. The results provide a description of the overall molecular shape and possible general three-dimensional modular arrangement for CCN proteins. These data unequivocally provide insight of the nature of CCN protein(s) in solution and thus important insight into their structure-function relationships.

Elastin enables the reversible deformation of elastic tissues and can withstand decades of repetitive forces. Tropoelastin is the soluble precursor to elastin, the main elastic protein found in mammals. Little is known of the shape and mechanism of assembly of tropoelastin as its unique composition and propensity to self-associate has hampered structural studies. In this study, we solve the nanostructure of full-length and corresponding overlapping fragments of tropoelastin using small angle X-ray and neutron scattering, allowing us to identify discrete regions of the molecule. Tropoelastin is an asymmetric coil, with a protruding foot that encompasses the C-terminal cell interaction motif. We show that individual tropoelastin molecules are highly extensible yet elastic without hysteresis to perform as highly efficient molecular nanosprings. Our findings shed light on how biology uses this single protein to build durable elastic structures that allow for cell attachment to an appended foot. We present a unique model for head-to-tail assembly which allows for the propagation of the molecule’s asymmetric coil through a stacked spring design.

Two-component regulatory systems mediate most of the bacterial cells responses to a variety of signals. In Sinorhizobium meliloti, the FixL-FixJ couple controls the expression of the nitrogen fixation genes through the binding of the two-domains response regulator FixJ to the fixK and nifA promoters. Phosphorylation of the N-terminal regulatory domain activates the protein and releases the inhibition of the C-terminal DNA-binding domain that occurs in the unphosphorylated protein. Insights into the transition from the inactive to the active form are provided by the architecture of the unphosphorylated response regulator reported in this study. The relative position and orientation of the N and C-terminal domains were defined from the molecular envelope restored from small-angle X-ray scattering (SAXS) data. The involvement of the alpha4-beta5-alpha5 surface of the regulatory domain, the linker region and the C-terminal helix of the DNA-binding domain in the interdomain interface of unphosphorylated FixJ was supported by biochemical investigations. These results, together with the previously reported studies on the phosphorylated regulatory domain of FixJ, emphasize the role of the alpha4-beta5-alpha5 surface in mediating a flow of information in this response regulator. This first study by SAXS of proteins from two-component systems suggests that the method could be successfully applied to other members of this family and could be suitable for the study of multidomain proteins and protein-protein complexes regulated through molecular interfaces in the low micromolar range.

Small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS) experiments were performed to investigate the micelle structure of n-octyl-β-maltopyranoside (OM). Density measurements were carried out to obtain the volumetric characteristics of OM for micelle structure analysis. Both temperature and concentration were varied in scattering experiments to study their effects on micelle size. The scattering data were analyzed by the indirect Fourier transformation method (IFT) and model fitting. The IFT method gave the radius of gyration of the micelles and their pair distance distribution function. It was found that the radii of gyration from SANS data were much smaller than those from SAXS data at similar solution conditions. Moreover, pair distance distribution functions from SANS and SAXS data were also different. Model fitting indicated that a spherical shell model can be used to describe both SANS and SAXS data using similar structure parameters. Comparison of SAXS data in D2O and H2O sho...

Several experimental methods, such as zeta potential, gel electrophoresis, small-angle X-ray scattering, gene transfection, fluorescence microscopy, flow cytometry, and cell viability/cytotoxicity assays, have been used to analyze the potential of anionic lipids (AL) as effective nontoxic and nonviral DNA vectors, assisted by divalent cations. The lipoplexes studied are those comprised of the green fluorescent protein-encoding plasmid DNA pEGFP-C3, an anionic lipid as 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG) or 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and a zwitterionic lipid, the 1,2-dioleoyl-sn -glycero-3-phosphatidylethanolamine (DOPE, not charged at physiological pH). The studies have been carried on at different liposome and lipoplex compositions and in the presence of a variety of [Ca2+]. Electrochemical experiments reveal that DOPG/DOPE and DOPS/DOPE anionic liposomes may compact more effectively pDNA at low molar fractions (with an excess of DOPE) and at AL/pDNA ratios ≈20. Calcium concentrations around 15-20 mM are needed to yield lipoplexes neutral or slightly positive. From a structural standpoint, DOPG/DOPE-Ca2+-pDNA lipoplexes are self-assembled into a HIIc phase (inverted cylindrical micelles in hexagonal ordering with plasmid supercoils inside the cylinders), while DOPS/DOPE-Ca2+-pDNA lipoplexes show two phases in coexistence: one classical HIIc phase which contains pDNA supercoils and one Lα phase without pDNA among the lamellae, i.e., a lamellar stack of lipidic bilayers held together by Ca2+ bridges. Transfection and cell viability studies were done with HEK293T and HeLa cells in the presence of serum. Lipoplexes herein studied show moderate-to-low transfection levels combined with moderate-to-high cell viability, comparable to those yield by Lipofectamine2000*, which is a cationic lipid (CL) standard formulation, but none of them improve the output of typical CL gen vectors, mostly if they are gemini or dendritic. This fact would be indicating that, nowadays, lipofection via anionic lipids and divalent cations as mediators still needs to enhance transfection levels in order to be considered as a real and plausible alternative to lipofection through improved CLs-based lipoplexes.

Neurodegenerative diseases constitute a large group of affections characterized by brain dysfunction and degeneration, often occurring with advancing age. Among them, prion diseases are fatal transmissible spongiform encephalopathies (TSEs) affecting Humans (Creutzfeldt-Jacob disease) and animals (BSE or the "Mad cow" disease). The causes of prion diseases have long been debated and it is widely accepted by the scientific community that a protein, namely, the prion protein (PrP), plays a critical role in the disease development. The prion protein or PrP is present in two structurally distinct forms: the natural cellular form (PrPc) and an abnormal polymeric form (PrPsc), which is at the origin of fibrillar deposits found in affected brains. PrPc has been shown to convert into small toxic oligomeric species but the mechanisms of oligomerization are not fully understood. In this study, we have combined Molecular Dynamics (MD) simulations and Small Angle X-ray Scattering (SAXS) to investigate the critical unfolding steps of the ovine prion protein (OvPrP) and to determine the low resolution structure of some of the oligomeric species obtained from various truncated forms of the OvPrP.We show that the domain formed by Helix2 and Helix3 (H2H3) is critical in the unfolding of OvPrP and its conversion of into a beta-rich conformer. The analysis also suggests the incorporation of 9-10 monomer units for all monomeric species studied. We therefore propose that the minimal β-rich core of OvPrP-O3 oligomer is formed by the H2H3 domain while the N-terminal domain either combines with other N-terminal domains to form a second domain or surrounds the oligomeric core.

The quaternary structures of monomeric and dimeric Drosophila non-claret disjunctional (ncd) constructs were investigated using synchrotron x-ray and neutron solution scattering, and their low resolution shapes were restored ab initio from the scattering data. The experimental curves were further compared with those computed from crystallographic models of one monomeric and three available dimeric ncd structures in the microtubule-independent ADP-bound state. These comparisons indicate that accounting for the missing parts in the crystal structures for all these constructs is indispensable to obtain reasonable fits to the scattering patterns. A ncd construct (MC6) lacking the coiled-coil region is monomeric in solution, but the calculated scattering from the crystallographic monomer yields a poor fit to the data. A tentative configuration of the missing C-terminal residues in the form of an antiparallel beta-sheet was found that significantly improves the fit. The atomic model of a short dimeric ncd construct (MC5) without 2-fold symmetry is found to fit the data better than the symmetric models. Addition of the C-terminal residues to both head domains gives an excellent fit to the x-ray and neutron experimental data, although the orientation of the beta-sheet differs from that of the monomer. The solution structure of the long ncd construct (MC1) including complete N-terminal coiled-coil and motor domains is modeled by adding a straight coiled-coil section to the model of MC5.

Data acquisition packages developed at different small angle scattering facilities use different formats both for raw and processed data storage. To facilitate the data exchange between laboratories, a consensus in the small angle scattering community has been reached on an ASCII format for one-dimensional data which includes a self-describing header containing relevant information about the sample and instrumental conditions followed by raw or reduced data in a tabular form. This format called sasCIF was implemented as an extension of core CIF (Crystallographic Information File) dictionary.

The sigma54 RNA polymerase holoenzyme functions in enhancer-dependent transcription. The structural organization of the sigma54 subunit of bacterial RNA polymerase in solution is analyzed by synchrotron x-ray scattering. Scattering patterns are collected from the full-length protein and from a large fragment able to bind the core RNA polymerase, and their low resolution shapes are restored using two ab initio shape determination techniques. The sigma54 subunit is a highly elongated particle, and the core binding fragment can be unambiguously positioned inside the full-length protein. The boomerang-like shape of the core binding fragment is similar to that of the atomic model of a fragment of the Escherichia coli sigma70 protein, indicating that, although the sigma54 and sigma70 factors are unrelated by primary sequence, they may share some structural similarity. Potential DNA binding surfaces of sigma54 are also predicted by comparison with the sigma54 core binding fragment.

New results obtained from a two-dimensional sequence analysis of the small heat shock protein (shsp) family are described. It is confirmed that the conserved C-terminal α -crystallin domain is essentially made of β -strands, most probably two groups of β -strands separated by a large loop. A direct correspondence between the putative β -strands that have been identified in shsps and the seven β -strands of a classical immunoglobulin-like fold is proposed. The hypothesis that the shsp family could belong to the immunoglobulin superfamily (IgSF) is consistent with the ubiquitous distribution and the multifunctional properties of the crystallins that are now emerging.

alphaB-crystallin, a member of the small heat-shock protein family and a major eye lens protein, is a high molecular mass assembly and can act as a molecular chaperone. We report a synchrotron radiation x-ray solution scattering study of a truncation mutant from the human alphaB-crystallin (alphaB57-157), a dimeric protein that comprises the alpha-crystallin domain of the alphaB-crystallin and retains a significant chaperone-like activity. According to the sequence analysis (more than 23% identity), the monomeric fold of the alpha-crystallin domain should be close to that of the small heat-shock protein from Methanococcus jannaschii (MjHSP16.5). The theoretical scattering pattern computed from the crystallographic model of the dimeric MjHSP16.5 deviates significantly from the experimental scattering by the alpha-crystallin domain, pointing to different quaternary structures of the two proteins. A rigid body modeling against the solution scattering data yields a model of the alpha-crystallin domain revealing a new dimerization interface. The latter consists of a strand-turn-strand motif contributed by each of the monomers, which form a four-stranded, antiparallel, intersubunit composite beta-sheet. This model agrees with the recent spin labeling results and suggests that the alphaB-crystallin is composed by flexible building units with an extended surface area. This flexibility may be important for biological activity and for the formation of alphaB-crystallin complexes of variable sizes and compositions.

International audience; Biological macromolecules in solution interact with each other through medium-range (from a few Angstrom to a few nm) interaction potentials. These potentials control the macromolecular distribution in solution, the macromolecular phase diagram and the crystallization process. We have previously shown that small angle X-ray scattering (SAXS) is a convenient tool to characterize the resulting potential, either attractive or repulsive, and to follow the changes induced by the crystallizing agents. In the present paper SAXS and simulation methods derived from statistical mechanics are coupled to determine the best fit potentials from the comparison of experimental and theoretical intensity curves. The currently used models in the colloid field are derived from the DLVO (Derjaguin, Landau, Verwey, Overbeek) potential where three types of interactions play a major role: hard sphere and electrostatic are repulsive, van der Waals are attractive. A combination of a short-range attractive potential and a coulombic repulsive indeed correctly accounts at low ionic strength for the phase diagram as a function of pH and salt concentration. The origin of the ion specificities at high ionic strength associated with the so-called "Hofmeister series" remain, however, unclear. The whole of the data demonstrates that the colloidal approach may be applied with success to protein crystallization. (C) 1999 Elsevier Science B.V. All rights reserved.