Arpan Nayak

A universal pathway for amyloid nucleus and precursor formation for insulin

To help identify the etiological agents for amyloid‐related diseases, attention is focused here on the fibrillar precursors, also called oligomers and protofibrils, and on modeling the reaction kinetics of the formation of the amyloid nucleus. Insulin is a favored model for amyloid formation, not only because amyloidosis can be a problem in diabetes, but also because aggregation and fibrillation causes problems during production, storage, and delivery. Small angle neutron scattering (SANS) is used to measure the temporal formation of insulin oligomers in H2O‐ and D2O‐based solvents and obtain consistent evidence of the composition of the insulin nucleus that comprised three dimers or six monomers similar to that recently proposed in the literature. A simple molecular structural model that describes the growth of oligomers under a wide range of environmental conditions is proposed. The model first involves lengthening or end‐on‐end association of dimers to form three‐dimer nuclei, and then exhibits broadening or side‐on‐side association of nuclei. Using different additives to demonstrate their influence on the kinetics of oligomer formation, we showed that, although the time required to form the nucleus was dependent on a specific system, they all followed a universal pathway for nucleus and precursor formation. The methods and analyses presented here provide the first experimental molecular size description of the details of amyloid nucleus formation and subsequent propagation to fibril precursors independent of kinetics. Proteins 2009.

Surface-enhanced nucleation of insulin amyloid fibrillation

Proteins can interact with biological surfaces such as cell membrane, chaperones, cornea, bone, arteries, veins, and heart cavities of the cardiovascular system and also with non-biological surfaces including dialysis membranes and tubing, catheters, invasive surgical instruments, needles, and artificial implants. Fibrillation of amyloid proteins is implicated in many human diseases, including Alzheimers, Parkinsons, and type II diabetes. Here, we show that heterogeneous surfaces accelerate the human insulin nucleation process that is the rate-determining step during amyloid fibril formation. The observed shorter lag (nucleation) phase correlates both with surface wettability and surface roughness. Surfaces promote faster nucleation possibly by increasing the local concentration of protein molecules. A composite parameter combining both surface wettability and roughness suggests that the ideal surface for slower nucleation should be hydrophilic and smooth. These findings provide a basis for designing suitable biomaterials and biomedical devices, especially those to resist amyloidosis.

Viscoelastic properties of adsorbed and cross-linked polypeptide and protein layers at a solid-liquid interface

The real-time changes in viscoelasticity of adsorbed poly(L-lysine) (PLL) and adsorbed histone (lysine rich fraction) due to cross-linking by glutaraldehyde and corresponding release of associated water were investigated using a quartz crystal microbalance with dissipation monitoring (QCM-D) and attenuated total reflection Fourier transform infrared spectroscopy (ATR/FTIR). The kinetics of PLL and histone adsorption were measured through changes in mass adsorbed onto a gold-coated quartz surface from changes in frequency and dissipation and using the Voigt viscoelastic model. Prior to cross-linking, the shear viscosity and shear modulus of the adsorbed PLL layer were approximately 3.0 x 10(-3) Pas and approximately 2.5 x 10(5) Pa, respectively, while after cross-linking, they increased to approximately 17.5 x 10(-3) Pas and approximately 2.5 x 10(6) Pa, respectively. For the adsorbed histone layer, shear viscosity and shear modulus increased modestly from approximately 1.3 x 10(-3) to approximately 2.0 x 10(-3) Pas and from approximately 1.2 x 10(4) to approximately 1.6 x 10(4) Pa, respectively. The adsorbed mass estimated from the Sauerbrey equation (perfectly elastic) and the Voigt viscoelastic model differ appreciably prior to cross-linking whereas after cross-linking they converged. This is because trapped water molecules were released during cross-linking. This was confirmed experimentally via ATR/FTIR measurements. The variation in viscoelastic properties increased substantially after cross-linking presumably due to fluctuation of the randomly cross-linked network structure. An increase in fluctuation of the viscoelastic properties and the loss of imbibed water could be used as a signature of the formation of a cross-linked network and the amount of cross-linking, respectively.

Osmolyte controlled fibrillation kinetics of insulin: New insight into fibrillation using the preferential exclusion principle

Amyloid proteins are converted from their native‐fold to long β‐sheet‐rich fibrils in a typical sigmoidal time‐dependent protein aggregation curve. This reaction process from monomer or dimer to oligomer to nuclei and then to fibrils is the subject of intense study. The main results of this work are based on the use of a well‐studied model amyloid protein, insulin, which has been used in vitro by others. Nine osmolyte molecules, added during the protein aggregation process for the production of amyloid fibrils, slow‐down or speed up the process depending on the molecular structure of each osmolyte. Of these, all stabilizing osmolytes (sugars) slow down the aggregation process in the following order: tri > di > monosaccharides, whereas destabilizing osmolytes (urea, guanidium hydrochloride) speed up the aggregation process in a predictable way that fits the trend of all osmolytes. With respect to kinetics, we illustrate, by adapting our earlier reaction model to the insulin system, that the intermediates (trimers, tetramers, pentamers, etc.) are at very low concentrations and that nucleation is orders of magnitude slower than fibril growth. The results are then collated into a cogent explanation using the preferential exclusion and accumulation of osmolytes away from and at the protein surface during nucleation, respectively. Both the heat of solution and the neutral molecular surface area of the osmolytes correlate linearly with two fitting parameters of the kinetic rate model, that is, the lag time and the nucleation rate prior to fibril formation. These kinetic and thermodynamic results support the preferential exclusion model and the existence of oligomers including nuclei and larger structures that could induce toxicity.

Egg White Varnishes on Ancient Paintings: A Molecular Connection to Amyloid Proteins

For about 400 years, egg white was used to coat and protect paintings without detailed understanding of its molecular properties. A molecular basis is provided for its advantageous properties and one of its protective properties is demonstrated with oxygen transport behavior. Compared to the native secondary structure of ovalbumin in solution of circa 33% α-helix and β-sheet, attenuated total reflection-FTIR (ATR-FTIR) spectra showed a 73% decrease of α-helix content and a 44% increase of β-sheet content over eight days. The data suggest that the final coating of dissolved ovalbumin from egg white after long exposure to air, which is hydrophobic, comprises mostly β-sheet content (ca. 50%), which is predicted to be the lowest-energy structure of proteins and close to that found in amyloid fibrils. Coating a synthetic polytetrafluoroethylene membrane with multiple layers of egg white decreased oxygen diffusion by 50% per layer with a total decrease of almost 100% for four layers.