Wojciech Dzwolak

Insulin forms amyloid in a strain‐dependent manner: An FT‐IR spectroscopic study

The presence of 20% (v/v) ethanol triggers growth of insulin amyloid with distinct infrared spectroscopic features, compared with the fibrils obtained under ambient conditions. Here we report that the two insulin amyloid types behave in the prion strain‐like manner regarding seeding specificity and ability of the self‐propagating conformational template to overrule unfavorable environmental factors and maintain the initial folding pattern. The type of the original seed has been shown to prevail over cosolvent effects and determines spectral position and width of the amide I′ infrared band of the heterogeneously seeded amyloid. These findings imply that “strains” may be a common generic trait of amyloids.

Fourier transform infrared spectroscopy in high-pressure studies on proteins

Several aspects of the application of Fourier transform infrared spectroscopy (FTIR) in high-pressure studies on proteins are reviewed. Basic methodological considerations regarding spectral band assignments, quantitative analysis, and choice of pressure calibrants are also placed within the scope of this paper. This work attempts to evaluate recent developments in the field of high-pressure FTIR of proteins and its prospects for future. Particular attention is paid to the phenomenon of protein aggregation.

Chiral bias of amyloid fibrils revealed by the twisted conformation of Thioflavin T: An induced circular dichroism/DFT study

Since it was implicated in a number of neurodegenerative conditions, such as Alzheimer disease, formation of β‐sheet‐rich protein fibrils (amyloids) has been drawing a lot of attention. One of elusive aspects of amyloidogenesis concerns the mechanisms of specific binding of molecules such as Congo red, or Thioflavin T by amyloid fibrils. A comprehensive understanding of these docking interactions is needed, however, for the sake of furthering biochemical studies and developing molecular, pharmacological strategies preventing proliferation of amyloids in vivo. Through the application of circular dichroism, here we show that upon binding to insulin fibrils, a twisted conformation is enforced in molecules of Thioflavin T, manifested in a strong negative Cotton effect around 450 nm, which is supported by density functional theory–based calculations. This finding may lead to circular dichroism of Thioflavin T becoming a new diagnostic technique for protein fibrils, complementary to fluorescence spectroscopy.

Hydration and structure—the two sides of the insulin aggregation process

Aggregation of bovine insulin under monomer, and dimer-promoting conditions has been probed by FT-IR spectroscopy and DSC/PPC calorimetry. This approach enabled linking of characteristic aggregation-hallmarking conformational events (such as partial unfolding, and subsequent refolding into aggregated β-strands) to different stages of protein hydration. Aggregation of insulin in 20% acetic acid occurs at a markedly lower temperature than in pure water and is further enhanced in the presence of charge-shielding ions (0.1 M NaCl). While acetic acid makes the protein molecule more permissive to the solvent penetration (seen as the enhanced H/D-exchange), NaCl does not have this effect. The calorimetric data reveals that, while the aggregation itself is exothermic, in the absence of the co-solvent it is preceded by an endothermic transition. In either case, the negative heat capacity changes suggest a substantial reduction in the number of water–protein contacts upon aggregation. This has been supported by pressure perturbation calorimetry, which showed a simultaneous release of water molecules bound to the protein. The aggregation of insulin appears to be driven mostly by hydrophobic interactions and, as such, involves a rearrangement of hydrophobic side-chain amino acid residues that leads to the overall reduction in number of unfavorable protein–water contacts. The study points to the intricate nature of the acetic acid effect on insulin aggregation, which is likely to involve changing the structure of the solvating water, and direct binding to the protein.

Noncooperative Dimethyl Sulfoxide-Induced Dissection of Insulin Fibrils: Toward Soluble Building Blocks of Amyloid

The enormous molecular weight complicates detailed structural studies of amyloid fibrils and obscures identification of biologically active forms of protein aggregates in amyloid-related diseases. Here we show that aqueous solutions of dimethyl sulfoxide (DMSO) solubilize insulin fibrils while maintaining their beta-pleated structure. This is accompanied by a marked decrease in the fluorescence of thioflavin T. According to atomic force microscopy images and dynamic light scattering measurements, the partial DMSO-induced dissection of insulin fibrils favors formation of smaller soluble oligomers, which retain a limited capacity to induce daughter generation of fibrils through seeding to the native insulin, as well as the ability to reassemble into fibrils upon removal of DMSO through dialysis against water. These findings suggest that the DMSO-induced ensembles of insulin molecules are closely related to elementary building blocks of amyloid fibrils.

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.

Size-dependent density of zirconia nanoparticles

Summary The correlation between density and specific surface area of ZrO2 nanoparticles (NPs) was studied. The NPs were produced using a hydrothermal process involving microwave heating. The material was annealed at 1100 °C which resulted in an increase in the average grain size of the ZrO2 NPs from 11 to 78 nm and a decrease in the specific surface area from 97 to 15 m2/g. At the same time, the density increased from 5.22 g/m3 to 5.87 g/m3. This effect was interpreted to be the result of the presence of a hydroxide monolayer on the NP surface. A smaller ZrO2 grain size was correlated with a larger contribution of the low density surface layer to the average density. To prove the existence of such a layer, the material was synthesized using 50% heavy water. Fourier transform infrared spectroscopy (FTIR) permitted the identification of the –OD groups created during synthesis. It was found that the –OD groups persisted on the ZrO2 surface even after annealing at 1100 °C. This hydroxide layer is responsible for the decrease in the average density of the NPs as their size decreases. This study of the correlation between particle size and density may be used to assess the quality of the NPs. In most cases, the technological aim is to avoid an amorphous layer and to obtain fully crystalline nanoparticles with the highest density possible. However, due to the effect of the surface layers, there is a maximum density which can be achieved for a given average NP diameter. The effect of the surface layer on the NP density becomes particularly evident for NPs smaller than 50 nm, and thus, the density of nanoparticles is size dependent.

Thioflavin T: Electronic Circular Dichroism and Circularly Polarized Luminescence Induced by Amyloid Fibrils

The circularly polarized luminescence (CPL) spectrum of thioflavin T (ThT) bound to insulin amyloid fibrils has been measured for the first time. It has been found that the samples exhibiting induced circular dichroism (CD) retain the optical activity in the CPL spectra, with the same sign of the rotatory strength. The fluorescence dissymmetry factor is substantial (of the order of magnitude 10(-2) ). Unlike in the corresponding CD and absorption spectra, there is no shift of the CPL band with respect to the fluorescence band. It has been verified that the measured CPL spectra are free from artifacts from circularly polarized scattering of emitted light by conducting additional measurements in a medium with a refractive index similar to insulin (methylsalicylate). The CD and CPL spectra have been interpreted by means of density functional calculations carried out for ThT in its ground and first excited states in different dielectric environments and for ThT interacting with an aromatic ring. It has been found that the presence of an aromatic ring close to the ThT molecule induces Cotton effects of the same order of magnitude as the stabilization of one enantiomeric conformer. Thus, it is expected that both mechanisms contribute to the induced CD and CPL effect to a similar degree.

Dimethyl Sulfoxide Induced Destabilization and Disassembly of Various Structural Variants of Insulin Fibrils Monitored by Vibrational Circular Dichroism

Dimethyl sulfoxide (DMSO) induced destabilization of insulin fibrils has been previously studied by Fourier transform infrared spectroscopy and interpreted in terms of secondary structural changes. The variation of this process for fibrils with different types of higher-order morphological structures remained unclear. Here, we utilize vibrational circular dichroism (VCD), which has been reported to provide a useful biophysical probe of the supramolecular chirality of amyloid fibrils, to characterize changes in the macroscopic chirality following DMSO-induced disassembly for two types of insulin fibrils formed under different conditions, at different reduced pH values with and without added salt and agitation. We confirm that very high concentrations of DMSO can disaggregate both types of insulin fibrils, which initially maintained a β-sheet conformation and eventually changed their secondary structure to a disordered form. The two types responded to varying concentrations of DMSO, and disaggregation followed different mechanisms. Interconversion of specific insulin fibril morphological types also occurred during the destabilization process as monitored by VCD. With transmission electron microscopy, we were able to correlate the changes in VCD sign patterns to alteration of morphology of the insulin fibrils.

Vortex-induced amyloid superstructures of insulin and its component A and B chains.

Insulin is an amyloid-forming polypeptide built of two disulfide-linked chains (A and B), both themselves amyloidogenic. An interesting property of insulin is that agitation strongly influences the course of its aggregation, resulting in characteristic chiral superstructures of amyloid fibrils. Here, we investigate the self-assembly of these superstructures by comparing the quiescent and vortex-assisted aggregation of insulin and its individual A and B chains in the presence or absence of reducing agent tris(2-carboxyethyl)phosphine (TCEP). Our study shows that only the B chain in the presence of TCEP is converted into aggregates with morphology (according to atomic force microscopy) and optical activity (manifested as an extrinsic Cotton effect induced in bound thioflavin T) characteristic of amyloid superstructures that are normally formed by insulin in the absence of TCEP. In contrast to more rigid B-peptide fibrils, elongated aggregates of the A peptide become amorphous upon agitation. Moreover, the aggregation of equimolar mixture of both peptides does not produce highly ordered entities. Our results suggest that the dynamics of the B chain are the driving force for the assembly of superstructures, with the A chain being complicit as long as its own dynamics are controlled by the firm attachment to the B chain provided by the intact covalent structure of insulin.