The degree of structural protection at the edge beta-strands determines the pathway of amyloid formation in globular proteins

The assembly of proteins into highly organized fibrillar aggregates is a key process in biology, biotechnology, and human disease. It has been shown that proteins retain a small, yet significant propensity to aggregate when they are folded into compact globular structures, and this may be physiologically relevant, particularly when considering that proteins spend most of their lifespan into such compact states. Proteins from the acylphosphatase-like structural family have been shown to aggregate via different mechanisms, with some members forming native-like aggregates as a first step of their aggregation process and others requiring unfolding as a first necessary step. Here we use the acylphosphatase from Sulfolobus solfataricus to show that assembly of folded protein molecules into native-like aggregates is prevented by single-point mutations that introduce structural protections within one of the most flexible region of the protein, the peripheral edge beta-strand 4. The resulting mutants do not form native-like aggregates, but can still form thioflavin T-binding and beta-structured oligomers, albeit more slowly than the wild-type protein. The kinetic data show that formation of the latter species proceeds via an alternative mechanism that is independent of the transient formation of native-like aggregates.     

On the structure formation of hydrophobed particles in the boundary layer of water and octane phases

Two-dimensional aggregation of the surface modified glass beads was carried out in the boundary layer of water and octane phases. The effect of particles' hydrophobicity was investigated on the structure of forming aggregates and the growth process. The structure of the aggregates and their growth were characterized by a density function which demonstrates the change of mean particle density as a function of aggregate size. The growth yielded fractal or nonfractal structures in the investigated systems. The fractal structure of the aggregates was observed to be dependent on restructuring processes controlled by the surface properties of the beads.The experimental results are compared with earlier findings for aggregation of hydrophobic beads in the boundary layer of water and air phases.On leave from Loránd Eötvös University, Budapest, Hungary     

Time evolution of the formation of different size cationic liposome-polyelectrolyte complexes

We report on the time evolution of the aggregation behaviour of cationic liposome-polyelectrolyte complexes studied by means of dynamic light scattering technique. Pure dioleoyltrimethilammoniumpropane (DOTAP) and mixed DOTAP-dipalmitoylphosphatidylcholine (DPPC) liposomes in polyacrylate sodium salt aqueous solutions in a wide concentration range have been investigated and the size and size distributions of the resulting aggregates evaluated from the intensity autocorrelation function of the scattered light. Under appropriate conditions, we found two discrete aggregation regimes, resulting in two different structural arrangements, whose time evolution depends on the charge ratio and the polyelectrolyte molecular weight. A first small component of average size in the 100-500 range nm coexists with a larger component, whose typical size increases with time, up to some micrometers. The cluster growth from a single liposome, 70 nm in diameter, to the formation of polymer-coated liposome aggregates has been briefly discussed in the light of steric stabilization of colloids. Moreover, it has been found that the kinetics of aggregation of the larger, time-dependent, component follows a dynamical scaling within the diffusion-limited cluster aggregation (DLCA) regime. The understanding of structures resulting from interactions between polyelectrolytes with oppositely charged liposomes may help towards formulation of "lipoplexes" (cationic lipid-DNA complexes) to use as non-viral gene carriers.     

Aggregation Patterns of Proteasome in Injured Neurons Induced by Transient Cerebral Ischemia

Proteasome activity reduction is an important pathological phenomenon, resulting in proteins aggregation and neuronal death in the injured neurons induced by transient ischemia. Our previous report showed that the trap of proteasome in the protein aggregates was a reason to lead to the reduction of proteasome activity. However, the patterns of proteasome entered into protein aggregates are not clear. In this study, we used a global ischemia model, Hematoxylin-Eosin staining, differential centrifuge, proteas...     

Patterned and controlled polyelectrolyte fractal growth and aggregations

Two-dimensional patterned and controlled polyelectrolyte aggregations (e.g., tree-like ramified structures) created by microcontact printing have been demonstrated and discussed. Polyelectrolyte-micropatterned aggregations on surfaces were controlled by the micropattern size and shape of PDMS stamps. The formation of aggregates was dependent on the ink and surface conditions, and the aggregates consisted of two distinct layers; strongly adsorbed, primary uniform layers and weakly adsorbed, secondary aggregation layers positioned on top of the primary layers. The adsorption of the primary layers was strong enough not to be washed away, while the aggregated secondary layers were easily removed by washing. The aggregation of secondary layers showed typical tree-like ramified structures of fractal growth and aggregation. Directional and confined stamping led to directing and confining the growth of the fractal polyelectrolyte clusters, respectively. The micropatterned primary uniform layers were not removed by extensive washing, and they were identified by selective nickel plating and charged particle selective adsorption in which the surface formed positive and negative micropatterns. These functional and patterned surfaces have great potentials for advanced devices and sensors.     

Fractal Heteroaggregation of Oppositely Charged Colloids

Floc structures resulting from selective heteroaggregation of positively and negatively charged colloids are investigated as a function of number ratio and shear conditions at pH 6. Negatively charged silica and positively charged alumina-coated silica undergo rapid aggregation due to attractive electrostatic interactions. At either extreme in number ratio, growth is terminated at an early stage, presumably because the aggregates acquire the same sign of charge, eliminating the driving force for further aggregation. For intermediate number ratios, extensive growth occurs, since the distribution of positive and negative charges is more uniform. Structure evolution of large heteroaggregates is assessed by static light scattering. Shear strongly influences the packing geometry and the tendency for the aggregates to undergo restructuring. At high shear (N(Re)>2000), heteroaggregates show relatively dense packing and do not restructure. Fractal dimension D(f) decreases from 2.64 to 2.26 as the number of positive particles is increased. At low shear (N(Re)<200), packing of the particles is more open and restructuring occurs. The lowest observed fractal dimension is 1.79. In the absence of applied shear, heteroaggregates with D(f)=1.79 rearrange to more compact structures with D(f)=1.88. Copyright 2000 Academic Press.     

Deposition of spherical particles onto cylindrical solid surfaces. I. Numerical simulations

The permeability of fractal porous aggregates with realistic three-dimensional structure is investigated theoretically using model aggregates composed of identical spherical primary particles. Synthetic aggregates are generated by several techniques, including a lattice-based method, simulation of aggregation by differential settling and turbulent shear, and the specification of simple cubic structures, resulting in aggregates characterized by the number of primary particles, solid fraction, characteristic radius, and fractal dimension. Stokesian dynamics is used to determine the total hydrodynamic force on and the distribution of velocity within an aggregate exposed to a uniform flow. The aggregate permeability is calculated by comparing these values with the total force and velocity distribution calculated from the Brinkman equation applied locally and to the entire aggregate using permeability expressions from the literature. The relationship between the aggregate permeability and solid fraction is found to be best predicted by permeability expressions based on cylindrical rather than spherical geometrical elements, the latter tending to underestimate the aggregate permeability significantly. The permeability expressions of Jackson and James or Davies provide good estimates of the force on and flow through porous aggregates of known structure. These relationships are used to identify a number of general characteristics of fractal aggregates.     

In situ measurements of the formation and morphology of intracellular β-amyloid fibrils by super-resolution fluorescence imaging

Misfolding and aggregation of peptides and proteins is a characteristic of many neurodegenerative disorders, including Alzheimer's disease (AD). In AD the β-amyloid peptide (Aβ) aggregates to form characteristic fibrillar structures, which are the deposits found as plaques in the brains of patients. We have used direct stochastic optical reconstruction microscopy, dSTORM, to probe the process of in situ Aβ aggregation and the morphology of the ensuing aggregates with a resolution better than 20 nm. We are able to distinguish different types of structures, including oligomeric assemblies and mature fibrils, and observe a number of morphological differences between the species formed in vitro and in vivo, which may be significant in the context of disease. Our data support the recent view that intracellular Aβ could be associated with Aβ pathogenicity in AD, although the major deposits are extracellular, and suggest that this approach will be widely applicable to studies of the molecular mechanisms of protein deposition diseases.     

Synchrotron X-ray scattering study of the mechanism of nanopore generation in nanoporous organosilicate thin films imprinted with a reactive six-armed porogen

In situ grazing incidence small-angle X-ray scattering analysis was successfully performed during the thermal processing of film blends of polymethylsilsesquioxane (PMSSQ) precursor and triethoxysilyl-terminated six-arm poly(epsilon-caprolactone) (mPCL6) porogen. In addition, thermogravimetric analysis of the films was carried out in a nitrogen atmosphere. These measurements provide important information about the structures of the blend films and of the resulting porous films. In particular, they are used in this paper to establish the mechanism of the formation of imprinted pores within the blend films. During the heating run, the sacrificial thermal degradation of the porogen component commenced at 320 degrees C, generating pores in the resulting cured PMSSQ films. Only very limited porogen aggregation occurred during the blend film formation process (spin-coating and subsequent drying), and these porogen aggregates were of relatively small size and narrow size distribution. The observed restriction of the formation of such porogen aggregates was found to result from the favorable hybridization reaction of the porogen's reactive end groups with the reactive functional groups of the PMSSQ precursor, which competes with aggregation via reaction between the porogen molecules. The average radius (or half-size) of the porogen aggregates was in the range 2.45-3.98 nm, depending on the porogen loading (10-40 wt %). The porogen aggregates retained their size and size distribution until thermal degradation, which resulted in the imprinting of nanopores in the cured PMSSQ films with size and size distribution corresponding to those of the porogen aggregates. The porosities of the resulting nanoporous films were in the range 12.4-41.7%, depending on the initial porogen loading.     

基于ThT荧光寿命检测蛋清溶菌酶蛋白寡聚体

采用时间分辨荧光技术, 检测了不同形态蛋白聚集体的荧光染料硫磺素T(ThT)荧光寿命. 利用蛋清溶菌酶体外制备了蛋白聚集体; 采用透射电子显微镜(TEM)及ThT稳态荧光检测了结合蛋白纤维生长的动力学曲线, 确定其形成寡聚体及纤维样聚集体的特征和时间. 通过时间相关单光子计数(TCSPC)技术测定了蛋清溶菌酶单体、 寡聚体和淀粉样纤维的ThT荧光寿命曲线, 并拟合、 计算其荧光寿命. 根据圆二色谱(CD)分析结果推测聚集体的结构不同导致其与ThT的结合状态不同, 从而影响ThT荧光寿命. 结果表明, 通过测定ThT荧光寿命可以区分蛋白单体、 寡聚体和纤维样聚集体, 并监测蛋白寡聚体的形成, 为后续病理蛋白聚集过程中形成寡聚体物质的监测提供了研究基础.     

Matrix Isolation-Vibrational Circular Dichroism Spectroscopic Study of Conformations and Non-Covalent Interactions of Tetrahydro-2-Furoic Acid

The conformational landscape and aggregation behaviour of tetrahydro-2-furoic acid (THFA) were investigated by using matrix isolation-vibrational circular dichroism (MI-VCD). The well-resolved experimental MI-IR and MI-VCD features in an argon matrix at 10 K allow one to identify two dominant monomeric conformations as trans-THFA where the hydroxyl and carbonyl groups of COOH are at opposite sides, as well as one cis-conformer. At 24 K and 30 K deposition temperatures, the experimental IR and VCD spectral features reveal further growth of the binary THFA aggregates. Systematic conformational searches identified three vastly different binary binding topologies, resulting in a few hundred stable (THFA)₂ conformers. Interestingly, the main binary structures observed correspond to an unusual type of structure which is made of two trans-THFA subunits, in contrast to the usual double H-bonded ring binary structures, identified in a previous solution study. The present work showcases the power of MI-VCD spectroscopy in revealing unusual structures formed in a cold rare gas matrix.     

In Silico Evidence That Protein Unfolding is a Precursor of Protein Aggregation

We present a computational study on the folding and aggregation of proteins in an aqueous environment, as a function of its concentration. We show how the increase of the concentration of individual protein species can induce a partial unfolding of the native conformation without the occurrence of aggregates. A further increment of the protein concentration results in the complete loss of the folded structures and induces the formation of protein aggregates. We discuss the effect of the protein interface on the water fluctuations in the protein hydration shell and their relevance in the protein-protein interaction.     

On the dynamics of the TPPS4 aggregation in aqueous solutions: successive formation of H and J aggregates

The dynamics of aggregation of meso-tetrakis (p-sulfonatofenyl) porphyrin (TPPS4) in function of its concentration, pH and ionic strength was studied by optical absorption, fluorescence and resonance light scattering (RLS) techniques. In the region of pH, where TPPS4 exists in biprotonated form, the addition of NaCl induces the TPPS4 aggregation due to the formation of the "cloud" of counter ions around the TPPS4 molecule thus reducing electrostatic repulsion between the porphyrin molecules. The formation of this "cloud" shifts the pKa value to acid region (from 5.0 in the absence of salt to 4.5 at [NaCl] = 0.4 M), reduces the TPPS4 absorption in all spectral range and quantum yield and lifetime of fluorescence (from 0.27 to 0.17 and from 4.00+/-0.04 to 3.00+/-0.03 ns in the absence of salt and in the presence of NaCl, respectively). The aggregation process involves two successive stages: initially H aggregates are formed, which in time are transformed in J ones. The existence of these two stages was confirmed by the fluorescence and RLS data. The kinetics of the formation of J aggregates is characterized by the induction time t1 and the average growth time t2, which depend on both TPPS4 and salt concentrations. The induction period t1 appears as a result of initial formation of H aggregates and their successive transformation in J ones. At very high TPPS4 concentrations, the J aggregates are united in more complex structures such as hollow cylinders or helixes.     

Effect of the Bulkiness of the End Functional Amide Groups on the Optical,Gelation, and Morphological Properties of Oligo(p‐phenylenevinylene) π‐Gelators

Herein, we describe the role of end functional groups in the self‐assembly of amide‐functionalized oligo(p‐phenylenevinylene) (OPV) gelators with different end‐groups. The interplay between hydrogen‐bonding and π‐stacking interactions was controlled by the bulkiness of the end functional groups, thereby resulting in aggregates of different types, which led to the gelation of a wide range of solvents. The variable‐temperature UV/Vis absorption and fluorescence spectroscopic features of gelators with small end‐groups revealed the formation of 1D H‐type aggregates in CHCl₃. However, under fast cooling in toluene, 1D H‐type aggregates were formed, whereas slow cooling resulted in 2D H‐type aggregates. OPV amide with bulky dendritic end‐group formed hydrogen‐bonded random aggregates in toluene and a morphology transition from vesicles into fibrous aggregates was observed in THF. Interestingly, the presence of bulky end‐group enhanced fluorescence in the xerogel state and aggregation in polar solvents. The difference between the aggregation properties of OPV amides with small and bulky end‐groups allowed the preparation of self‐assembled structures with distinct morphological and optical features.     

Spectroscopic, morphological, and mechanistic investigation of the solvent-promoted aggregation of porphyrins modified in meso-positions by glucosylated steroids

Solvent-driven aggregation of a series of porphyrin derivatives was studied by UV/Vis and circular dichroism spectroscopy. The porphyrins are characterised by the presence in the meso positions of steroidal moieties further conjugated with glucosyl groups. The presence of these groups makes the investigated macrocycles amphiphilic and soluble in aqueous solvent, namely, dimethyl acetamide/water. Aggregation of the macrocycles is triggered by a change in bulk solvent composition leading to formation of large architectures that express supramolecular chirality, steered by the presence of the stereogenic centres on the periphery of the macrocycles. The aggregation behaviour and chiroptical features of the aggregates are strongly dependent on the number of moieties decorating the periphery of the porphyrin framework. In particular, experimental evidence indicates that the structure of the steroid linker dictates the overall chirality of the supramolecular architectures. Moreover, the porphyrin concentration strongly affects the aggregation mechanism and the CD intensities of the spectra. Notably, AFM investigations reveal strong differences in aggregate morphology that are dependent on the nature of the appended functional groups, and closely in line with the changes in aggregation mechanism. The suprastructures formed at lower concentration show a network of long fibrous structures spanning over tens of micrometres, whereas the aggregates formed at higher concentration have smaller rod-shaped structures that can be recognised as the result of coalescence of smaller globular structures. The fully steroid substituted derivative forms globular structures over the whole concentration range explored. Finally, a rationale for the aggregation phenomena was given by semiempirical calculations at the PM6 level.     

Single-Molecule Two-Color Coincidence Detection of Unlabeled alpha-Synuclein Aggregates

Protein misfolding and aggregation into oligomeric and fibrillar structures is a common feature of many neurogenerative disorders. Single-molecule techniques have enabled characterization of these lowly abundant, highly heterogeneous protein aggregates, previously inaccessible using ensemble averaging techniques. However, they usually rely on the use of recombinantly-expressed labeled protein, or on the addition of amyloid stains that are not protein-specific. To circumvent these challenges, we have made use of a high affinity antibody labeled with orthogonal fluorophores combined with fast-flow microfluidics and single-molecule confocal microscopy to specifically detect α-synuclein, the protein associated with Parkinson's disease. We used this approach to determine the number and size of α-synuclein aggregates down to picomolar concentrations in biologically relevant samples.