Measurement of length distribution of beta-lactoglobulin fibrils by multiwavelength analytical ultracentrifugation

The whey protein beta-lactoglobulin is the building block of amyloid fibrils which exhibit a great potential in various applications. These include stabilization of gels or emulsions. During biotechnological processing, high shear forces lead to fragmentation of fibrils and therefore to smaller fibril lengths. To provide insight into such processes, pure straight amyloid fibril dispersions (prepared at pH 2) were produced and sheared using the rotor stator setup of an Ultra Turrax. In the first part of this work, the sedimentation properties of fragmented amyloid fibrils sheared at different stress levels were analyzed with mulitwavelength analytical ultracentrifugation (AUC). Sedimentation data analysis was carried out with the boundary condition that fragmented fibrils were of cylindrical shape, for which frictional properties are known. These results were compared with complementary atomic force microscopy (AFM) measurements. We demonstrate how the sedimentation coefficient distribution from AUC experiments is influenced by the underlying length and diameter distribution of amyloid fibrils. In the second part of this work, we show how to correlate the fibril size reduction kinetics with the applied rotor revolution and the resulting energy density, respectively, using modal values of the sedimentation coefficients obtained from AUC. Remarkably, the determined scaling laws for the size reduction are in agreement with the results for other material systems, such as emulsification processes or the size reduction of graphene oxide sheets.</p

Towards recombinantly produced milk proteins: Physicochemical and emulsifying properties of engineered whey protein beta-lactoglobulin variants

DFG, 273937032, SPP 1934: Dispersitäts-, Struktur- und Phasenänderungen von Proteinen und biologischen Agglomeraten in biotechnologischen ProzessenBMBF, 031B0222, Basistechnologie Nachwuchsgruppe "Multiskalige Modellierung und Modifikation von Multienzymkomplexen als Basistechnologie für zellfreie Reaktionskaskaden" (II

Whey protein (amyloid)-aggregates in oil-water systems : The process-related comminution effect

Whey protein fibrils are excellent emulsifiers. However, the emulsification process significantly alters the size of the aggregates and thus their functionality. It is not yet known how strongly the disperse phase (oil) contributes to the size change. Furthermore, it is unknown whether the aggregate morphologies (semi-flexible amyloid-, and flexible amyloid-like aggregates) differ in their size reduction during emulsification. Therefore, both types of aggregates were processed under different stress levels and in the presence and absence of an oil phase by rotor-stator dispersion, ultrasonication and high-pressure homogenisation. The size reduction exponent for each aggregate type was determined by atomic force microscopy, analytical ultracentrifugation and dynamic light scattering. Semi-flexible fibrils decreased in length by rotor-stator shear from 6200 to a minimum of 190 nm, but sonication resulted in even greater shortening (150 - 84 nm) and is comparable to high pressure homogenisation (283 - 111 nm). Worm-like flexible aggregates are only affected by sonication (98 - 46 nm). The addition of oil resulted in a further reduced aggregate length with lower energy input for all aggregates. Overall, these results provide new insights about the emulsion processing behaviour of different amyloid aggregates, which should be taken into account when preparing emulsions

The threshold of amyloid aggregation of beta-lactoglobulin : Relevant factor combinations

This study identifies critical factor combinations of pH, temperature, stirring speed, protein and ion concentration that specifically affect the lag-, and growth phase of beta-lactoglobulin amyloid aggregation and provides information on how, when and why certain factor combinations affect the onset of amyloid aggregation. Conditions at the threshold of amyloid formation were chosen to prolong the lag and growth phase for several hours. Temperature was the most important factor in all aggregation stages. Interactions between low pH and temperature in the growth phase were caused by elevated protein denaturation at low pH (DSC). Interactions between stirring speed and temperature in the lag phase were caused by viscosity-dependent shear stress (CFD-simulation). Even if none of the factor combinations could shift the onset of amyloid aggregation, important factor combinations were identified that favour the onset of amyloid aggregation. Therefore, the results of this study could be relevant for industrial production.</p

Toxic Metamorphosis—How Changes from Lysosomal to Cytosolic pH Modify the Alpha-Synuclein Aggregation Pattern

Alpha-synuclein (aSyn) is a cytosolic, aggregation-prone protein that is associated with neurodegenerative disorders like Parkinson’s disease. Interestingly, the protein can appear in different conformations, including monomeric and oligomeric forms as well as amyloid fibrils. Its individual structural constituents seem to be dependent on various factors and the composition of the respective cellular surroundings. Although under physiological conditions, most aSyn is found in the cytosol and synapses of neurons, aSyn can also be found in lysosomal compartments, where it gets degraded. We here compare the assembly speed, morphology, folding state, and spreading of aSyn at cytosolic pH (pH 7.4) and lysosomal pH (pH 5) using Thioflavin T, transmission electron microscopy, circular dichroism, and Fourier transform infrared spectroscopy. Interestingly, we found substantial differences between aSyn aggregation under neutral and acidic pH conditions, like those present in cytosolic and lysosomal cellular compartments. Also, lysosomal aSyn enriched from an aSyn-overexpressing cell line was able to seed aggregation in a concentration-dependent manner. Moreover, we observed that aSyn aggregates formed under in vitro lysosomal pH (pH 5) conditions were not stable at neutral pH and collapsed into partly soluble aggregates with changed structural characteristics. Our findings have meaningful implications in intracellular toxicity events as well as in lysis procedures for molecular and structural characterization of intracellular aSyn conformers.<br/

Influence of the polydispersity of pH 2 and pH 3.5 beta-lactoglobulin amyloid fibril solutions on analytical methods

It is well known that amyloid beta-lactoglobulin (BLG) fibril solutions contain a heterogeneous mixture of amyloid aggregates and non-amyloid material. However, few information are available on how strongly separated fractions of different morphologies (straight fibrils at pH 2 and worm-like aggregates at pH 3.5) vary with respect to physicochemical properties and building blocks as most analyses are conducted with unfractionized solutions where superposition effects occur. The pH-value shift resulted in an altered degree of acid hydrolysis which led to dissimilar building blocks of the aggregates (peptides at pH 2, non-hydrolyzed protein at pH 3.5). The respective separated amyloid and non-amyloid fractions showed significantly different size (SAXS, SEC, AUC) and charge properties (Zeta potential) than the whole samples. Strong superposition effects were evident with common analyses such as FTIR, TRP fluorescence and Thioflavin-T. At the same time, structural differences of pH 2 and pH 3.5 aggregates could be presented more clearly.</p