Functional diversity of YbbN/CnoX proteins: Insights from a comparative analysis of three thioredoxin-like oxidoreductases from Pseudomonas aeruginosa, Xylella fastidiosa and Escherichia coli.

YbbN/CnoX are proteins that display a Thioredoxin (Trx) domain linked to a tetratricopeptide domain. YbbN from Escherichia coli (EcYbbN) displays a co-chaperone (holdase) activity that is induced by HOCl. Here, we compared EcYbbN with YbbN proteins from Xylella fastidiosa (XfYbbN) and from Pseudomonas aeruginosa (PaYbbN). EcYbbN presents a redox active Cys residue at Trx domain (Cys63), 24 residues away from SQHC motif (SQHC[N24]C) that can form mixed disulfides with target proteins. In contrast, XfYbbN and PaYbbN present two Cys residues in the CXXC (CAPC) motif, while only PaYbbN shows the Cys residue equivalent to Cys63 of EcYbbN. Our phylogenetic analysis revealed that most of the YbbN proteins are in the bacteria domain of life and that their members can be divided into four groups according to the conserved Cys residues. EcYbbN (SQHC[N24]C), XfYbbN (CAPC[N24]V) and PaYbbN (CAPC[N24]C) are representatives of three sub-families. In contrast to EcYbbN, both XfYbbN and PaYbbN: (1) reduced an artificial disulfide (DTNB) and (2) supported the peroxidase activity of Peroxiredoxin Q from X. fastidiosa, suggesting that these proteins might function similarly to the canonical Trx enzymes. Indeed, XfYbbN was reduced by XfTrx reductase with a high catalytic efficiency (kcat/Km = 1.27 x 107 M-1 s-1), similar to the canonical XfTrx (XfTsnC). Furthermore, EcYbbN and XfYbbN, but not PaYbbN displayed HOCl-induced holdase activity. Remarkably, EcYbbN gained disulfide reductase activity while lost the HOCl-activated chaperone function, when the SQHC was replaced by CQHC. In contrast, the XfYbbN CAPA mutant lost the disulfide reductase activity, while kept its HOCl-induced chaperone function. In all cases, the induction of the holdase activity was accompanied by YbbN oligomerization. Finally, we showed that deletion of ybbN gene did not render in P. aeruginosa more sensitive stressful treatments. Therefore, YbbN/CnoX proteins display distinct properties, depending on the presence of the three conserved Cys residues.

Assessing the Structure and Equilibrium Conditions of Complex Coacervate Core Micelles by Varying Their Shell Composition and Medium Ionic Strength.

Complex coacervates result from an associative phase separation commonly involving oppositely charged polyelectrolytes. When this associative interaction occurs between charged-neutral diblock copolymers and oppositely charged homopolymers, a nanometric aggregate called a complex coacervate core micelle, C3M, is formed. Recent studies have addressed the issue of their thermodynamic or kinetic stability but without a clear consensus. To further investigate this issue, we have studied C3Ms formed by the combination of poly(diallyldimethylammonium) and copolymer poly(acrylamide)-b-poly(acrylate) using different preparation protocols. Dynamic light scattering and small-angle X-ray scattering measurements suggest that these structures are in an equilibrium condition because the aggregates do not vary with different preparation protocols or upon aging. In addition, their stability and structures are critically dependent on several parameters such as the density of neutral blocks in their shell and the ionic strength of the medium. Decreasing the amount of copolymer in the system and, hence, the density of neutral blocks in the shell results in an increase in the aggregate size because of the core growth, although their globular shape is retained. On the other hand, larger clusters of micelles were formed at higher ionic strengths. Partially replacing 77% of the copolymer with a homopolymer of the same charge or increasing the ionic strength of the system (above 100 mmol L-1 NaCl) leads to a metastable state, after which phase separation is eventually observed. SAXS analyses reveal that this phase separation above a certain salt concentration occurs due to the coagulation of individual micelles that seem to retain their individual globular structures. Overall, these results confirm earlier claims that equilibrium C3Ms are achieved close to 1:1 charge stoichiometry but also reveal that these conditions may vary at different shell densities or higher ionic strengths, which constitute vital information for envisioning future applications of C3Ms.

Glucose restriction in Saccharomyces cerevisiae modulates the phosphorylation pattern of the 20S proteasome and increases its activity.

Caloric restriction is known to extend the lifespan and/or improve diverse physiological parameters in a vast array of organisms. In the yeast Saccharomyces cerevisiae, caloric restriction is performed by reducing the glucose concentration in the culture medium, a condition previously associated with increased chronological lifespan and 20S proteasome activity in cell extracts, which was not due to increased proteasome amounts in restricted cells. Herein, we sought to investigate the mechanisms through which glucose restriction improved proteasome activity and whether these activity changes were associated with modifications in the particle conformation. We show that glucose restriction increases the ability of 20S proteasomes, isolated from Saccharomyces cerevisiae cells, to degrade model substrates and whole proteins. In addition, threonine 55 and/or serine 56 of the α5-subunit, were/was consistently found to be phosphorylated in proteasomes isolated from glucose restricted cells, which may be involved in the increased proteolysis capacity of proteasomes from restricted cells. We were not able to observe changes in the gate opening nor in the spatial conformation in 20S proteasome particles isolated from glucose restricted cells, suggesting that the changes in activity were not accompanied by large conformational alterations in the 20S proteasome but involved allosteric activation of proteasome catalytic site.

Retrieving the size distribution of SBA-15 mesopores from small-angle X-ray scattering data using a Monte Carlo method.

A Monte Carlo (MC) method was introduced into a state-of-the-art model used to analyse small-angle X-ray scattering (SAXS) data of SBA-15, an ordered mesoporous material with many applications. With this new procedure, referred to herein as the SBA-15+MC model, it is possible to retrieve the size distribution of the mesopores, D(r), in a free modelling approach. To achieve this, two main points were addressed: (i) based on previous implementations, the method was adapted to work with long core-shell cylinders; (ii) since the MC model requires longer processing times, strategies to speed up the calculations were developed, which included a simplified version of the original model used to analyse SAXS data of SBA-15 (referred to as the SBA-15 model) as well as the determination of several structural features from the SAXS curve prior to the fit. The new model was validated with simulated data and later used to fit experimental SAXS curves of SBA-15. The obtained results show that the SBA-15 model only works well because the mesopore size distribution of SBA-15 is narrow, whereas the new approach can be successfully used in cases where D(r) is wider and/or has a more complex profile, such as SBA-15 with expanded mesopores. Even though a specific SAXS example was chosen to prove the model, the strategies presented herein are general and suitable for inclusion in other models aimed at the analysis of SBA-15 and similar ordered mesoporous materials.

New Lyotropic Complex Fluid Structured in Sheets of Ellipsoidal Micelles Solubilizing Fragrance Oils.

New lyotropic, fragranced, viscoelastic fluid with a complex structure is obtained from fragranced microemulsions by the addition of a fatty acid. Nonhomogeneous mixing of an appropriate nonionic surfactant, a fatty acid, and a fragrance oil led to the formation of anisotropically shaped and highly oriented micelles in aqueous solution. The nano- and microstructures, and consequently the viscosity, are controlled by the balance of fatty acids used as a cosurfactant and fragrance molecules, which partly behave as a cosurfactant and partly segregate in the micelles of the hydrophilic nonionic surfactant. The transition from isotropic microemulsion to a more structured viscoelastic solution is characterized by X-ray scattering and rheological methods. Considering our X-ray scattering results, we propose a structure composed of planar sheets of ellipsoidal micelles arranged in a lamellar type of stacking. The complex structured, low viscous, transparent fluid is capable of solubilizing a fragrance inside the ellipsoidal micelles, as well as retaining microparticles containing fragrance, without the addition of a polymeric thickener or another gelator. These features allow the creation of a 2-in-1 fragrance-solubilizing liquid product compatible with all types of home and body care consumer products.

Glucose restriction in Saccharomyces cerevisiae modulates the phosphorylation pattern of the 20S proteasome and increases its activity

Caloric restriction is known to extend the lifespan and/or improve diverse physiological parameters in a vast array of organisms. In the yeast Saccharomyces cerevisiae, caloric restriction is performed by reducing the glucose concentration in the culture medium, a condition previously associated with increased chronological lifespan and 20S proteasome activity in cell extracts, which was not due to increased proteasome amounts in restricted cells. Herein, we sought to investigate the mechanisms through which glucose restriction improved proteasome activity and whether these activity changes were associated with modifications in the particle conformation. We show that glucose restriction increases the ability of 20S proteasomes, isolated from Saccharomyces cerevisiae cells, to degrade model substrates and whole proteins. In addition, threonine 55 and/or serine 56 of the α5-subunit, were/was consistently found to be phosphorylated in proteasomes isolated from glucose restricted cells, which may be involved in the increased proteolysis capacity of proteasomes from restricted cells. We were not able to observe changes in the gate opening nor in the spatial conformation in 20S proteasome particles isolated from glucose restricted cells, suggesting that the changes in activity were not accompanied by large conformational alterations in the 20S proteasome but involved allosteric activation of proteasome catalytic site.

Adjuvant effect of mesoporous silica SBA-15 on anti-diphtheria and anti-tetanus humoral immune response.

Routine immunization against diphtheria and tetanus has drastically reduced the incidence of these diseases worldwide. Anti-diphtheria/tetanus vaccine has in general aluminum salt as adjuvant in its formulation that can produce several adverse effects. There is a growing interest in developing new adjuvants. In this study, we evaluated the efficiency of SBA-15 as an adjuvant in subcutaneous immunization in mice with diphtheria (dANA) and tetanus (tANA) anatoxins as well as with the mixture of them (dtANA). The tANA molecules and their encapsulation in SBA-15 were characterized using Small-Angle X-ray Scattering (SAXS), Dynamical Light Scattering (DLS), Nitrogen Adsorption Isotherm (NAI), Conventional Circular Dichroism (CD)/Synchrotron Radiation Circular Dichroism (SRCD) Spectroscopy, and Tryptophan Fluorescence Spectroscopy (FS). The primary and secondary antibody response elicited by subcutaneous immunization of High (HIII) and Low (LIII) antibody responder mice with dANA, tANA, or dtANA encapsulated in the SBA-15 were determined. We demonstrated that SBA-15 increases the immunogenicity of dANA and tANA antigens, especially when administered in combination. We also verified that SBA-15 modulates the antibody response of LIII mice, turning them into high antibody responder. Thus, these results suggest that SBA-15 may be an effective adjuvant for different vaccine formulations.

Staphylococcus aureus Exfoliative Toxin E, Oligomeric State and Flip of P186: Implications for Its Action Mechanism.

Staphylococcal exfoliative toxins (ETs) are glutamyl endopeptidases that specifically cleave the Glu381-Gly382 bond in the ectodomains of desmoglein 1 (Dsg1) via complex action mechanisms. To date, four ETs have been identified in different Staphylococcus aureus strains and ETE is the most recently characterized. The unusual properties of ETs have been attributed to a unique structural feature, i.e., the 180° flip of the carbonyl oxygen (O) of the nonconserved residue 192/186 (ETA/ETE numbering), not conducive to the oxyanion hole formation. We report the crystal structure of ETE determined at 1.61 Å resolution, in which P186(O) adopts two conformations displaying a 180° rotation. This finding, together with free energy calculations, supports the existence of a dynamic transition between the conformations under the tested conditions. Moreover, enzymatic assays showed no significant differences in the esterolytic efficiency of ETE and ETE/P186G, a mutant predicted to possess a functional oxyanion hole, thus downplaying the influence of the flip on the activity. Finally, we observed the formation of ETE homodimers in solution and the predicted homodimeric structure revealed the participation of a characteristic nonconserved loop in the interface and the partial occlusion of the protein active site, suggesting that monomerization is required for enzymatic activity.

Influence of the Mixtures of Vegetable Oil and Vitamin E over the Microstructure and Rheology of Organogels.

Candelilla wax (CW) and 12-hydroxystearic acid (12HSA) are classic solid-fiber-matrix organogelators. Despite the high number of studies using those ingredients in oily systems, there is scarce literature using a mixture of oil and antioxidants. Vitamin E (VE) is an important candidate for its lipophilicity and several applications on pharmaceutical, cosmetics, and food industries. In this work, we investigated the influences of mixtures between vegetable oil (VO) and VE on the microstructures and rheological properties of CW and 12HSA organogels. A weak gel (G''/G' > 0.1) with a shear-thinning behavior was observed for all samples. The presence of VE impacted the gel strength and the phase transition temperatures in a dose-dependent pattern. Larger and denser packed crystals were seen for 12HSA samples, while smaller and more dispersed structures were obtained for CW organogels. The results obtained in this work allowed the correlation of the structural and mechanical properties of the organogels, which plays an important role in the physical-chemical characteristics of these materials.

Using crystallography tools to improve vaccine formulations.

This article summarizes developments attained in oral vaccine formulations based on the encapsulation of antigen proteins inside porous silica matrices. These vaccine vehicles show great efficacy in protecting the proteins from the harsh acidic stomach medium, allowing the Peyer's patches in the small intestine to be reached and consequently enhancing immunity. Focusing on the pioneering research conducted at the Butantan Institute in Brazil, the optimization of the antigen encapsulation yield is reported, as well as their distribution inside the meso- and macroporous network of the porous silica. As the development of vaccines requires proper inclusion of antigens in the antibody cells, X-ray crystallography is one of the most commonly used techniques to unveil the structure of antibody-combining sites with protein antigens. Thus structural characterization and modelling of pure antigen structures, showing different dimensions, as well as their complexes, such as silica with encapsulated hepatitis B virus-like particles and diphtheria anatoxin, were performed using small-angle X-ray scattering, X-ray absorption spectroscopy, X-ray phase contrast tomography, and neutron and X-ray imaging. By combining crystallography with dynamic light scattering and transmission electron microscopy, a clearer picture of the proposed vaccine complexes is shown. Additionally, the stability of the immunogenic complex at different pH values and temperatures was checked and the efficacy of the proposed oral immunogenic complex was demonstrated. The latter was obtained by comparing the antibodies in mice with variable high and low antibody responses.

Using crystallography tools to improve vaccine formulations

This article summarizes developments attained in oral vaccine formulations based on the encapsulation of antigen proteins inside porous silica matrices. These vaccine vehicles show great efficacy in protecting the proteins from the harsh acidic stomach medium, allowing the Peyer's patches in the small intestine to be reached and consequently enhancing immunity. Focusing on the pioneering research conducted at the Butantan Institute in Brazil, the optimization of the antigen encapsulation yield is reported, as well as their distribution inside the meso- and macroporous network of the porous silica. As the development of vaccines requires proper inclusion of antigens in the antibody cells, X-ray crystallography is one of the most commonly used techniques to unveil the structure of antibody-combining sites with protein antigens. Thus structural characterization and modelling of pure antigen structures, showing different dimensions, as well as their complexes, such as silica with encapsulated hepatitis B virus-like particles and diphtheria anatoxin, were performed using small-angle X-ray scattering, X-ray absorption spectroscopy, X-ray phase contrast tomography, and neutron and X-ray imaging. By combining crystallography with dynamic light scattering and transmission electron microscopy, a clearer picture of the proposed vaccine complexes is shown. Additionally, the stability of the immunogenic complex at different pH values and temperatures was checked and the efficacy of the proposed oral immunogenic complex was demonstrated. The latter was obtained by comparing the antibodies in mice with variable high and low antibody responses.

Structure and Thermotropic Behavior of Bovine- and Porcine-Derived Exogenous Lung Surfactants.

Two commercial exogenous pulmonary surfactants, Curosurf and Survanta, are investigated. Their thermotropic behavior and associated structural changes for the samples in bulk are characterized and described. For Survanta, the obtained results of differential scanning calorimetry showed a thermogram with three peaks on heating and only a single peak on cooling. Curosurf on the other hand, presents calorimetric thermograms with only one peak in both the heating and cooling scans. This distinct thermotropic behavior between the two pulmonary surfactants, a consequence of their particular compositions, is associated with structural changes that were evaluated by simultaneous small- and wide-angle X-ray scattering experiments with in situ temperature variation. Interestingly, for temperatures below ∼35 °C for Curosurf and ∼53 °C for Survanta, the scattering data indicated the coexistence of two lamellar phases with different carbon chain organizations. For temperatures above these limits, the coexistence of phases disappears, giving rise to a fluid phase in both pulmonary surfactants, with multilamelar vesicles for Curosurf and unilamellar vesicles for Survanta. This process is quasi-reversible under cooling, and advanced data analysis for the scattering data indicated differences in the structural and elastic properties of the pulmonary surfactants. The detailed and systematic investigation shown in this work expands on the knowledge of the structure and thermodynamic behavior of Curosurf and Survanta, being relevant from both physiological and biophysical perspectives and also providing a basis for further studies on other types of pulmonary surfactants.

Proinflammatory Action of a New Electronegative Low-Density Lipoprotein Epitope.

The electronegative low-density lipoprotein, LDL (-), is an endogenously modified LDL subfraction with cytotoxic and proinflammatory actions on endothelial cells, monocytes, and macrophages contributing to the progression of atherosclerosis. In this study, epitopes of LDL (-) were mapped using a phage display library of peptides and monoclonal antibodies reactive to this modified lipoprotein. Two different peptide libraries (X6 and CX8C for 6- and 8-amino acid-long peptides, respectively) were used in the mapping. Among all tested peptides, two circular peptides, P1A3 and P2C7, were selected based on their high affinities for the monoclonal antibodies. Small-angle X-ray scattering analysis confirmed their structures as circular rings. P1A3 or P2C7 were quickly internalized by bone marrow-derived murine macrophages as shown by confocal microscopy. P2C7 increased the expression of TNFα, IL-1 ß and iNOS as well as the secretion of TNFα, CCL2, and nitric oxide by murine macrophages, similar to the responses induced by LDL (-), although less intense. In contrast, P1A3 did not show pro-inflammatory effects. We identified a mimetic epitope associated with LDL (-), the P2C7 circular peptide, that activates macrophages. Our data suggest that this conformational epitope represents an important danger-associated molecular pattern of LDL (-) that triggers proinflammatory responses.

Nanoscopic Structure of Complexes Formed between DNA and the Cell-Penetrating Peptide Penetratin.

One of the most remarkable examples of cell-penetrating peptides (CPPs) is Penetratin, a 16-mer fragment derived from the Drosophila Antennapedia homeobox. Understanding the structure of Penetratin/DNA complexes is a key factor for the successful design of new vectors for gene delivery and may assist in optimizing molecular carriers based on CPPs. Herein, we present a comprehensive study on the nanoscale structure of noncovalent complexes formed between Penetratin and DNA. The strong cationic nature of the peptide makes it a very efficient agent for condensing DNA strands via electrostatic attraction, and we show for the first time that DNA condensation is accompanied by random-to-ß-sheet transitions of Penetratin secondary structure, demonstrating that nucleic acids behave as a structuring agent upon complexation. For the first time, nanoscale-resolved spectroscopy is used to provide single-particle infrared data from DNA carriers based on CPPs, and they show that the structures are stabilized by Penetratin ß-sheet cores, whereas larger DNA fractions are preferentially located in the periphery of aggregates. In-solution infrared assays indicate that phosphate diester groups are strongly affected upon DNA condensation, presumably as a consequence of charge delocalization induced by the proximity of cationic amide groups in Penetratin. The morphology is characterized by nanoassemblies with surface fractal features, and short-range order is found in the inner structure of the scaffolds. Interestingly, the formation of beads-on-a-string arrays is found, producing nanoscale architectures that resemble structures observed in early steps of chromatin condensation. A complexation pathway where DNA condensation and peptide pairing into ß-sheets are key steps for organization is proposed.

Structure of the haptoglobin-haemoglobin complex.

Red cell haemoglobin is the fundamental oxygen-transporting molecule in blood, but also a potentially tissue-damaging compound owing to its highly reactive haem groups. During intravascular haemolysis, such as in malaria and haemoglobinopathies, haemoglobin is released into the plasma, where it is captured by the protective acute-phase protein haptoglobin. This leads to formation of the haptoglobin-haemoglobin complex, which represents a virtually irreversible non-covalent protein-protein interaction. Here we present the crystal structure of the dimeric porcine haptoglobin-haemoglobin complex determined at 2.9 Å resolution. This structure reveals that haptoglobin molecules dimerize through an unexpected ß-strand swap between two complement control protein (CCP) domains, defining a new fusion CCP domain structure. The haptoglobin serine protease domain forms extensive interactions with both the α- and ß-subunits of haemoglobin, explaining the tight binding between haptoglobin and haemoglobin. The haemoglobin-interacting region in the αß dimer is highly overlapping with the interface between the two αß dimers that constitute the native haemoglobin tetramer. Several haemoglobin residues prone to oxidative modification after exposure to haem-induced reactive oxygen species are buried in the haptoglobin-haemoglobin interface, thus showing a direct protective role of haptoglobin. The haptoglobin loop previously shown to be essential for binding of haptoglobin-haemoglobin to the macrophage scavenger receptor CD163 (ref. 3) protrudes from the surface of the distal end of the complex, adjacent to the associated haemoglobin α-subunit. Small-angle X-ray scattering measurements of human haptoglobin-haemoglobin bound to the ligand-binding fragment of CD163 confirm receptor binding in this area, and show that the rigid dimeric complex can bind two receptors. Such receptor cross-linkage may facilitate scavenging and explain the increased functional affinity of multimeric haptoglobin-haemoglobin for CD163 (ref. 4).

Structure and dimerization of translation initiation factor aIF5B in solution.

Translation initiation factor 5B (IF5B) is required for initiation of protein synthesis. The solution structure of archaeal IF5B (aIF5B) was analysed by small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) and was indicated to be in both monomeric and dimeric form. Sedimentation equilibrium (SE) analytical ultracentrifugation (AUC) of aIF5B indicated that aIF5B forms irreversible dimers in solution but only to a maximum of 5.0-6.8% dimer. Sedimentation velocity (SV) AUC at higher speed also indicated the presence of two species, and the sedimentation coefficients s(20,w)(0) were determined to be 3.64 and 5.51±0.29 S for monomer and dimer, respectively. The atomic resolution (crystallographic) structure of aIF5B (Roll-Mecak et al. [6]) was used to model monomer and dimer, and theoretical sedimentation coefficients for these models were computed (3.89 and 5.63 S, respectively) in good agreement with the sedimentation coefficients obtained from SV analysis. Thus, the structure of aIF5B in solution must be very similar to the atomic resolution structure of aIF5B. SAXS data were acquired in the same buffer with the addition of 2% glycerol to inhibit dimerization, and the resultant monomeric aIF5B in solution did indeed adopt a structure very similar to the one reported earlier for the protein in crystalline form. The p(r) function indicated an elongated conformation supported by a radius of gyration of 37.5±0.2 Å and a maximum dimension of ~130 Å. The effects of glycerol on the formation of dimers are discussed. This new model of aIF5B in solution shows that there are universal structural differences between aIF5B and the homologous protein IF2 from Escherichia coli.

Structural transitions of translation initiation factor IF2 upon GDPNP and GDP binding in solution.

Three protein factors ensure rapid and accurate initiation of translation in bacteria. Translation initiation factor IF2 is a ribosome-dependent GTPase, which is important for correct positioning of initiator tRNA on the 30S subunit as well as ribosomal subunit joining. The solution structure of the free C-terminal part of IF2 (IF2C, comprising domains IV to VI-2) was previously determined by small-angle X-ray scattering (SAXS) [Rasmussen, L. C., et al. (2008) Biochemistry 47, 5590-5598]. In this study, adding GDP or nonhydrolyzable GTP analogue GDPNP to the protein in solution caused structural changes in the protein, in agreement with recent data determined via isothermal titration calorimetry [Hauryliuk, V., et al. (2009) J. Mol. Biol. 394, 621-626]. The p(r) function indicated an elongated conformation supported by radii of gyration of 40.1 and 44.9 Å and maximum dimensions of ~125 and ~150 Å for IF2C with GDPNP and GDP, respectively. The SAXS data were used to model the structure of IF2C bound to either GDPNP or GDP. The structural transitions of IF2C upon GDPNP binding and following nucleotide hydrolysis support the concept of cofactor-dependent conformational switching rather than the classical model for GTPase activity.

SDS-induced fibrillation of alpha-synuclein: an alternative fibrillation pathway.

A structural investigation of the sodium dodecyl sulfate (SDS)-induced fibrillation of alpha-synuclein (alphaSN), a 140-amino-acid protein implicated in Parkinson's disease, has been performed. Spectroscopic analysis has been combined with isothermal titration calorimetry, small-angle X-ray scattering, and transmission electron microscopy to elucidate a fibrillation pathway that is remarkably different from the fibrillation pathway in the absence of SDS. Fibrillation occurs most extensively and most rapidly (starting within 45 min) under conditions where 12 SDS molecules are bound per alphaSN molecule, which is also the range where SDS binding is associated with the highest enthalpy. Fibrillation is only reduced in proportion to the fraction of SDS below 25 mol% SDS in mixed surfactant mixtures with nonionic surfactants and is inhibited by formation of bulk micelles and induction of alpha-helical structure. In this fibrillogenic complex, 4 alphaSN molecules initially associate with 40-50 SDS molecules to form a shared micelle that gradually grows in size. The complex initially exhibits a mixture of random coil and alpha-helix, but incubation results in a structural conversion into beta-sheet structure and concomitant formation of thioflavin-T-binding fibrils over a period of several hours. Based on small-angle X-ray scattering, the aggregates elongate as a beads-on-a-string structure in which individual units of ellipsoidal SDS-alphaSN are bridged by strings of the protein, so that aggregates nucleate around the surface of protein-stabilized micelles. Thus, fibrillation in this case occurs by a process of continuous accretion rather than by the rate-limiting accumulation of a distinct nucleus. The morphology of the SDS-induced fibrils does not exhibit the classical rod-like structures formed by alphaSN when aggregated by agitation in the absence of SDS. The SDS-induced fibrils have a flexible worm-like appearance, which can be converted into classical straight fibrils by continuous agitation. SDS-induced fibrillation represents an alternative and highly reproducible mechanism for fibrillation where protein association is driven by the formation of shared micelles, which subsequently allows the formation of beta-sheet structures that presumably link individual micelles. This illustrates that protein fibrillation may occur by remarkably different mechanisms, testifying to the versatility of this process.

Structure of nanoscale truncated octahedral DNA cages: variation of single-stranded linker regions and influence on assembly yields.

The assembly, structure, and stability of DNA nanocages with the shape of truncated octahedra have been studied. The cages are composed of 12 double-stranded B-DNA helices interrupted by single-stranded linkers of thymidines of varying length that constitute the truncated corners of the structure. The structures assemble with a high efficiency in a one-step procedure, compared to previously published structures of similar complexity. The structures of the cages were determined by small-angle X-ray scattering. With increasing linker length, there is a systematic increase of the cage size and decrease of the twist angle of the double helices with respect to the symmetry planes of the cage structure. In the present study, we demonstrate the length of the single-stranded linker regions, which impose a certain degree of flexibility to the structure, to be the important determinant for efficient assembly. The linker length can be decreased to three thymidines without affecting assembly yield or the overall structural characteristics of the DNA cages. A linker length of two thymidines represents a sharp cutoff abolishing cage assembly. This is supported by energy minimization calculations suggesting substantial hydrogen bond deformation in a cage with linkers of two thymidines.

Polypeptide nanoribbon hydrogels assembled through multiple supramolecular interactions.

We investigated the formation of nanoribbon hydrogels in a mixed system of zinc ions, bis(ligand)s, and triblock peptide copolymers. Using a combination of experimental techniques: dynamic light scattering, cryo-transmission electron microscopy, small-angle X-ray scattering and circular dichroism, we arrived at a model for the formation of nanoribbon hydrogels in which well-defined nanoribbons are formed out of multiple supramolecular interactions: (1) metal coordination that yields supramolecular polyelectrolytes; (2) electrostatic complexation between the supramolecular polyelectrolytes and the oppositely charged blocks of the peptide copolymers; (3) hydrogen bond and (4) hydrophobic interactions that support the secondary and ternary structure of the ribbons; (5) van der Waals interactions that enable bundling of the ribbons.