Fibrillar β-Lactoglobulin Gels: Part 1. Fibril Formation and Structure

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Abstract
As a prelude to experimental and theoretical work on the mechanical properties of fibrillar β-lactoglobulin gels, this paper reports the structural characterization of β-lactoglobulin fibrils by electron and atomic force microscopy (AFM), infrared and Raman spectroscopy, and powder X-ray diffraction. Aggregates formed by incubation of β-lactoglobulin in various alcohol-water mixtures at pH 2, and in water-trifluoroethanol (TFE) at pH 7, were found to be wormlike (∼7 nm in width and 1 μm in length), smoother, and seemingly stiffer fibrils formed on heating aqueous β-lactoglobulin solutions at pH 2 and low ionic strength, although there was little evidence for the higher-order structures common in most amyloid-forming systems. Time-lapse AFM also revealed differences in the formation of these two fibril types: thermally induced aggregation occurring more cooperatively, in keeping with a nucleation and growth process. Only short stiff-rods (<20 nm in length) formed on heating β-lactoglobulin at pH 7, and only complex three-dimensional “amorphous” aggregates in alcohols other than TFE at this pH. Studies of all of the pH 2 fibrils from β-lactoglobulin, by Raman and infrared spectroscopy confirmed β-sheet as mediating the aggregation process. Interestingly, however, some evidence for de novo helix formation for the solvent-induced systems was obtained, although it remains to be seen whether this is actually incorporated into the fibril-structure. In contrast to other amyloid systems, X-ray powder diffraction provided no evidence for extensive repeating “crystalline” structures for any of the pH 2 β-lactoglobulin fibrils. In relation to amyloid, the lactoglobulin fibrils bear more resemblance to protofilaments than to higher-order fibril structures, these latter appearing more convincingly for thermally induced insulin fibrils (pH 2) also included in the AFM study.