Template-directed self-assembly and growth of insulin amyloid fibrils

The formation of amyloid aggregates in tissue is a pathological feature of many neurodegenerative diseases and type II diabetes. Amyloid deposition, the process of amyloid growth by the association of individual soluble amyloid molecules with a pre-existing amyloid template (i.e., plaque), is known to be critical for amyloid formation in vivo. The requirement for a natural amyloid template, however, has made amyloid deposition study difficult and cumbersome. In the present work, we developed a novel, synthetic amyloid template by attaching amyloid seeds covalently onto an N-hydroxysuccinimide-activated surface, where insulin was chosen as a model amyloidogenic protein. According to ex situ atomic force microscopy observations, insulin monomers in solution were deposited onto the synthetic amyloid template to form fibrils, like hair growth. The fibril formation on the template occurred without lag time, and its rate was highly accelerated than in the solution. The fibrils were long, over 2 mum, and much thinner than those in the solution, which was caused by limited nucleation sites on the template surface and lack of lateral twisting between fibrils. According to our investigations using thioflavin T-induced fluorescence, birefringent Congo red binding, and circular dichroism, fibrils grown on the template were identified to be amyloids that formed through a conformational rearrangement of insulin monomers upon interaction with the template. The amyloid deposition rate followed saturation kinetics with respect to insulin concentration in the solution. The characteristics of amyloid deposition on the synthetic template were in agreement with previous studies performed with human amyloid plaques. It is demonstrated that the synthetic amyloid template can be used for the screening of inhibitors on amyloid deposition in vitro.     

Nucleation and growth of microtubules from gamma-tubulin-functionalized gold surfaces

Microtubules are protein filaments that are emerging as potential building blocks in manufacturing nanoscale structures and systems such as interconnecting nanowires. Future development in using microtubules necessitates a control of their nucleation and growth. We report the controlled nucleation and growth of microtubules from functionalized gold on a hydrophilic oxidized silicon wafer. The gold substrate is functionalized with gamma-tubulin, a natural nucleating agent for microtubule growth. We show that the attached gamma-tubulin retains its biological functionality and leads to nucleation and assembly of microtubules from the functionalized gold surface. We also analyze the interplay between the geometry of the nucleating substrates and the morphology of microtubules arrays and networks grown from them. We consider two geometrical arrangements of the substrates: (a) a square lattice of small gold pads on a hydrophilic oxidized silicon wafer and (b) a large flat surface. Fluorescence microscopy and scanning electron microscopy are employed to provide a detailed characterization of the length and morphology of the nucleated and grown microtubules. The observed microtubule morphologies are modeled, analyzed and discussed within the context of reaction-diffusion and nucleation controlled processes.     

DnaK prevents human insulin amyloid fiber formation on hydrophobic surfaces

We have developed a multiwell-based protein aggregation assay to study the kinetics of insulin adsorption and aggregation on hydrophobic surfaces and to investigate the molecular mechanisms involved. Protein-surface interaction progresses in two phases: (1) a lag phase during which proteins adsorb and prefibrillar aggregates form on the material surface and (2) a growth phase during which amyloid fibers form and then are progressively released into solution. We studied the effect of three bacterial chaperones, DnaK, DnaJ, and ClpB, on insulin aggregation kinetics. In the presence of ATP, the simultaneous presence of DnaK, DnaJ, and ClpB allows good protection of insulin against aggregation. In the absence of ATP, DnaK alone is able to prevent insulin aggregation. Furthermore, DnaK binds to insulin adsorbed on hydrophobic surfaces. This process is slowed in the presence of ATP and can be enhanced by the cochaperone DnaJ. The peptide LVEALYL, derived from the insulin B chain, is known to promote fast aggregation in a concentration- and pH-dependent manner in solution. We show that it also shortens the lag phase for insulin aggregation on hydrophobic surfaces. As this peptide is also a known DnaK substrate, our data indicate that the peptide and the chaperone might compete for a common site during the process of insulin aggregation on hydrophobic surfaces.     

Chemical modification of insulin in amyloid fibrils

We have investigated the chemical modification of insulin under conditions that promote the conversion of the soluble protein into amyloid fibrils. The modifications that are incorporated into the fibrils include deamidation of Asn A21, Asn B3, and Gln B4. In order to prepare fibrils with minimal deamidation of these residues, the kinetics of aggregation were accelerated by seeding with aliquots of a solution containing preformed fibrils. The resulting fibrils were then reincubated to determine the extent to which chemical modification occurs in the fibril itself. The deamidation of Asn A21 in particular could be followed in detail. Deamidation of this residue in the fibrillar form of insulin was found to occur in only 52 +/- 5% of molecules. This result indicates that there are at least two different packing environments of insulin molecules in the fibrils and suggests that the characterization of chemical modifications may be a useful probe of the environment of polypeptide chains within amyloid fibrils.     

Adsorption behavior of a human monoclonal antibody at hydrophilic and hydrophobic surfaces

One aspiration for the formulation of human monoclonal antibodies (mAb) is to reach high solution concentrations without compromising stability. Protein surface activity leading to instability is well known, but our understanding of mAb adsorption to the solid-liquid interface in relevant pH and surfactant conditions is incomplete. To investigate these conditions, we used total internal reflection fluorescence (TIRF) and neutron reflectometry (NR). The mAb tested (“mAb-1”) showed highest surface loading to silica at pH 7.4 (~12 mg/m²), with lower surface loading at pH 5.5 (~5.5 mg/m², further from its pI of 8.99) and to hydrophobized silica (~2 mg/m²). The extent of desorption of mAb-1 from silica or hydrophobized silica was related to the relative affinity of polysorbate 20 or 80 for the same surface. mAb-1 adsorbed to silica on co-injection with polysorbate (above its critical micelle concentration) and also to silica pre-coated with polysorbate. A bilayer model was developed from NR data for mAb-1 at concentrations of 50–5000 mg/L, pH 5.5, and 50–2000 mg/L, pH 7.4. The inner mAb-1 layer was adsorbed to the SiO₂ surface at near saturation with an end-on” orientation, while the outer mAb-1 layer was sparse and molecules had a “side-on” orientation. A non-uniform triple layer was observed at 5000 mg/L, pH 7.4, suggesting mAb-1 adsorbed to the SiO₂ surface as oligomers at this concentration and pH. mAb-1 adsorbed as a sparse monolayer to hydrophobized silica, with a layer thickness increasing with bulk concentration - suggesting a near end-on orientation without observable relaxation-unfolding.     

Chiral superstructures of insulin amyloid fibrils

Hydrodynamic forces are capable of inducing structural order in dispersed solid phases, and of causing symmetry-breaking when chiral crystals precipitate from an achiral liquid phase. Until it was observed upon vortex-assisted fibrillation of insulin, such behavior had been thought to be confined to few unbiological systems. In this paper we are discussing chiroptical properties of two chiral variants of insulin amyloid, termed +ICD and -ICD, which form during the process of chiral bifurcation in vortexed solutions of aggregating insulin. As conventional measurements of circular dichroism of solid, anisotropic substances are particularly vulnerable to overlapping influences of linear birefringence and linear dichroism, we have employed complementary tools including dedicated universal chiroptical spectrophotometer to rule out such artifacts. We propose that the strong chiroptical properties of +ICD and -ICD insulin fibrils are an aspect of genuine superstructural chirality of amyloid fibrils and of powerful excitonic couplings taking place within them. A comparison of thioflavin T complexes with fibrils formed by insulin and polyglutamic acid suggests that the extrinsic Cotton effect stemming from the level of single twisted dye molecules is weaker, although diagnostically useful, and cannot account for the overall magnitude of ICD of the dye bound to ±ICD insulin amyloid.     

Morphological studies of oligodeoxyribonucleotides probes covalently immobilized at polystyrene modified surfaces

The immobilization of short ss-DNA (18- and 36-mer) and their hybridization were studied at gold and glassy carbon substrates modified with low molecular weight (approximately 12, 18 and 24 kg/mol) polystyrene thin films. Amino-modified DNA was attached to the surface by reaction with succinimide ester groups bound to the polystyrenes. A ferrocene modified DNA target was used to confirm the probe-target hybridization. Atomic force microscopy studies showed significant morphological changes after probe immobilization and hybridization compared to the featureless structure of the polystyrene film. Single-stranded DNA samples had a globular morphology with an average density of 3.8 and 2.2 (x 10(11)) globules/cm2 for the 18- and 36-mer, respectively. The formation of a porous structure with a 2.0 and 1.0 (x10(11)) average pore density corresponding to the 18- and 36-mer was observed after hybridization. A surface composition analysis was done by X-ray photoelectron spectroscopy to confirm and support the images interpretation. Ferrocene oxidation (+323 mV/18-mer, +367 mV/36-mer, versus Ag/AgCl) proved the presence of ds-DNA at the modified surfaces.     

Intravital study of the hydrophobic interactions in protein fibrils by a polarization-fluorescence method. I. Collagen fibrils

It has been shown earlier that anisotropy of extrinsic fluorescence (AEF) of ordered structures in living cell may serve as a measure of the free structure energy. The experimental study of AEF changes of myelin at varying temperatures (0-30 degrees C) revealed cold denaturation. This phenomenon occurs only in cases when the structure in question is stabilized through hydrophobic interactions. The purpose of this work was to show that not only membranes but also some native ordered protein structures might be analysed by fluorescence polarization to detect hydrophobic interactions. The fluorescence anisotropy of primulin adsorbed by collagen fibrils from rat tail has been investigated. The fluorophore orientation decreases with temperature, i.e. here the phenomenon of cold denaturation is observed. When the medium humidity falls down to 80%, no cold denaturation occurs. AEF of collagen fibrils depends to a considerable extent on organic substances (ethanol, ethylene glycol) added in small concentrations to the incubation medium. It is concluded that the dependence of the whole collagen molecular structure in fibrils on the content and structure of the solvent shell is of critical nature.     

Adhesion of cells to polystyrene surfaces

The surface treatment of polystyrene, which is required to make polystyrene suitable for cell adhesion and spreading, was investigated. Examination of surfaces treated with sulfuric acid or various oxidizing agents using (a) x-ray photoelectron and attenuated total reflection spectroscopy and (b) measurement of surface carboxyl-, hydroxyl-, and sulfur-containing groups by various radiochemical methods showed that sulfuric acid produces an insignificant number of sulfonic acid groups on polystyrene. This technique together with various oxidation techniques that render surfaces suitable for cell culture generated high surface densities of hydroxyl groups. The importance of surface hydroxyl groups for the adhesion of baby hamster kidney cells or leukocytes was demonstrated by the inhibition of adhesion when these groups were blocked: blocking of carboxyl groups did not inhibit adhesion and may raise the adhesion of a surface. These results applied to cell adhesion in the presence and absence of serum. The relative unimportance of fibronectin for the adhesion and spreading of baby hamster kidney cells to hydroxyl-rich surfaces was concluded when cells spread on such surfaces after protein synthesis was inhibited with cycloheximide, fibronectin was removed by trypsinization, and trypsin activity was stopped with leupeptin.     

Binding mode of Thioflavin T in insulin amyloid fibrils

Amyloid fibrils share various common structural features and their presence can be detected by Thioflavin T (ThT). In this paper, the binding mode of ThT to insulin amyloid fibrils was examined. Scatchard analysis and isothermal titration calorimetry (ITC) showed at least two binding site populations. The binding site population with the strongest binding was responsible for the characteristic ThT fluorescence. This binding had a capacity of about 0.1 moles of ThT bound per mole of insulin in fibril form. The binding capacity was unaffected by pH, but the affinity was lowest at low pH. Notably, presence of a third binding process prior to the other processes was suggested by ITC. Binding of ThT resulted in only minor changes in the fibril structure according to the X-ray diffraction patterns, where a slightly more dominant equatorial reflection at 16A relative to the intersheet distance of 11A was observed. No change in the interstrand distance of 4.8A was observed. On the basis of our results, we propose that ThT binds in cavities running parallel to the fibril axis, e.g., between the protofilaments forming the fibrils. Such cavities have been proposed previously in insulin fibrils and several other amyloid fibril models.     

Nucleation and growth of cadherin adhesions

Cell-cell contact formation relies on the recruitment of cadherin molecules and their anchoring to actin. However, the precise chronology of events from initial cadherin trans-interactions to adhesion strengthening is unclear, in part due to the lack of access to the distribution of cadherins within adhesion zones. Using N-cadherin expressing cells interacting with N-cadherin coated surfaces, we characterized the formation of cadherin adhesions at the ventral cell surface. TIRF and RIC microscopies revealed streak-like accumulations of cadherin along actin fibers. FRAP analysis indicated that engaged cadherins display a slow turnover at equilibrium, compatible with a continuous addition and removal of cadherin molecules within the adhesive contact. Association of cadherin cytoplasmic tail to actin as well as actin cables and myosin II activity are required for the formation and maintenance of cadherin adhesions. Using time lapse microscopy we deciphered how cadherin adhesions form and grow. As lamellipodia protrude, cadherin foci stochastically formed a few microns away from the cell margin. Neo-formed foci coalesced aligned and coalesced with preformed foci either by rearward sliding or gap filling to form cadherin adhesions. Foci experienced collapse at the rear of cadherin adhesions. Based on these results, we present a model for the nucleation, directional growth and shrinkage of cadherin adhesions.     

Analysis of insulin amyloid fibrils by Raman spectroscopy

The formation of amyloid fibrils from insulin is investigated using drop-coating-deposition-Raman (DCDR) difference spectroscopy and atomic force microscopy (AFM). Fibrils formed using various co-solvents and heating cycles are found to induce the appearance of Raman difference peaks in the amide I (approximately 1675 cm(-1)), amide III (approximately 1220 cm(-1)), and peptide backbone (approximately 1010 cm(-1)), consistent with an increase in beta-sheet content. Comparisons of results obtained from fibrils in either H2O or D2O suggest that the NH/ND stretch bands (at approximately 3300 cm(-1)/ approximately 2400 cm(-1)) are also enhanced in intensity upon fibril formation. If there is any water trapped in the core of the fibrils its OH/OD Raman intensity is too small to be detected in the presence of the stronger NH/ND bands which appear in the same region. AFM is used to confirm the formation of fibrils of about 5 nm diameter (and various lengths).     

Effects of confinement on insulin amyloid fibrils formation

Insulin, a 51-residue protein universally used in diabetes treatment, is known to produce amyloid fibrils at high temperature and acidic conditions. As for other amyloidogenic proteins, the mechanisms leading to nucleation and growth of insulin fibrils are still poorly understood. We here report a study of the fibrillation process for insulin confined in a suitable polymeric hydrogel, with the aim of ascertain the effects of a reduced protein mobility on the various phases of the process. The results indicate that, with respect to standard aqueous solutions, the fibrillation process is considerably slowed down at moderately high concentrations and entirely suppressed at low concentration. Moreover, the analysis of the initial stages of the fibrillation process in aqueous solutions revealed a large spatial heterogeneity, which is completely absent when the fibrillation is carried out in the hydrogel. We attribute this heterogeneity to the diffusion in solution of large amyloidal aggregates, which must be formed very fast compared to the average times for the whole sample. These findings are interpreted in the framework of recently suggested heterogeneous nucleation mechanisms. Moreover, they may be useful for the development of new insulin pharmaceutical formulations, more stable against adverse conditions.     

Higher-order molecular packing in amyloid-like fibrils constructed with linear arrangements of hydrophobic and hydrogen-bonding side-chains

Various mutants of the protein fragment, barnase module-1 (1-24) were investigated in order to reveal the structural principle of amyloid-like fibrils. By means of circular dichroism spectroscopy, X-ray diffraction, electron microscopy, and thioflavin T binding assay, we found that the molecules containing two beta-strands and an intervening turn structure are assembled to form a cross-beta structure. Stabilization by both the hydrophobic interactions and hydrogen bonding between the respective paired side-chains on the coupled beta-strands was essential for fibril formation. These two types of interaction can also arrange the corresponding residues in lines on both sheet surfaces of protofilaments with a cross-beta structure. This leads to the most probable fibril structure constructed with the line-matching interactions between protofilaments. Consideration of the geometrical symmetry resulted in our finding that a limited number of essential models for molecular packing in fibril structure are stable, which would rationally explain the occurrence of two or three morphologies from an identical molecular species. The ribbon-like fibrils exhibited striped texture along the axis, which was assigned to a stacked two-sheet repeat as a structural unit. The comprehensively proposed structural model, that is, the sheet-sheet interaction between left-handed cross-beta structures, results in a slightly right-handed twist of beta-sheet stacking, which reasonably elucidates the intrinsic sizes of the fibril width and its helical period along the fibril axis, as the bias in the orientation of the hydrogen-bonded beta-strand pair at the lateral edge is larger than that at the central protofilament.     

Investigation of the kinetics of insulin amyloid fibrils formation

Today, the investigation of the structure of ordered protein aggregates-amyloid fibrils, the influence of the native structure of the protein and the external conditions on the process of fibrillation-is the subject of intense investigations. The aim of the present work is to study the kinetics of formation of insulin amyloid fibrils at low pH values (conditions that are used at many stages of the isolation and purification of the protein) using the fluorescent probe thioflavin T. It is shown that the increase of the fluorescence intensity of ThT during the formation of amyloid fibrils is described by a sigmoidal curve, in which three areas can be distinguished: the lag phase, growth, and a plateau, which characterize the various stages of fibril formation. Despite the variation in the length of the lag phase at the same experimental conditions (pH and temperature), it is seen to drop during solution stirring and seeding. Data obtained by electron microscopy showed that the formed fibrils are long, linear filaments ～20 nm in diameter. With increasing incubation time, the fibril diameter does not change, while the length increases to 2–3 μm, which is accompanied by a significant increase in the number of fibril aggregates. All the experimental data show that, irrespective of the kinetics of formation of amyloid fibrils, their properties after the completion of the fibrillation process are identical. The results of this work, together with the previous studies of insulin amyloid fibrils, may be important for clarification the mechanism of their formation, as well as for the treatment of amyloidosis associated with the aggregation of insulin.     

Nucleation and growth of ice in deeply undercooled erythrocytes

Previous studies of the mechanism of freezing of erythrocytes in the absence of intracellular ice have been extended to define the catalytic sites responsible for promoting nucleation. The following aspects have been investigated: (1) the freeze propagation between undercooled erythrocytes, (2) the nucleation of ice in undercooled erythrocyte ghosts, and (3) the freezing behavior of undercooled hemoglobin solutions. The main findings are: (1) no cross-nucleation occurs between individual cells packed within the same emulsified water droplet; (2) the differential scanning calorimetric power-time curves of intact cells and ghosts are identical, indicating that hemoglobin does not affect ice nucleation; (3) the nucleation temperature of ice in an aqueous solution of hemoglobin (isolated from the cells) is substantially lower than that for the same solution when contained in the intact cell; (4) the threefold freeze concentration which accompanies the freezing of a 25% hemoglobin solution does not cause denaturation of the protein.     

Intravital research on hydrophobic interactions in protein fibrils using a fluorescence polarization method

Fluorescent probe of acridine orange (AO) is known to be bound exclusively by materials of thick protofibril muscle fibres. The dependence of fluorescence anisotropy of AO in frog muscle fibres on the temperature has been studied. It has been found that the fluorophore orientation decreases with temperature, i.e. here the adsorbent cold denaturation takes place. This phenomenon was demonstrated for living, skinned and glycerinated muscle fibres. It shows that hydrophobic interactions contribute much to stabilization of the myosin structure. After contraction of glycerinated muscle fibres at the action of ATP, the AO fluorescence anisotropy grows independently of the temperature which is indicative of structural rearrangements in materials of thick protofibrils upon contraction.