Fibrillar beta-lactoglobulin gels: Part 1. Fibril formation and structure.
As a prelude to experimental and theoretical work on the mechanical properties of fibrillar beta-lactoglobulin gels, this paper reports the structural characterization of beta-lactoglobulin fibrils by electron and atomic force microscopy (AFM), infrared and Raman spectroscopy, and powder X-ray diffraction. Aggregates formed by incubation of beta-lactoglobulin in various alcohol-water mixtures at pH 2, and in water-trifluoroethanol (TFE) at pH 7, were found to be wormlike (approximately 7 nm in width and <500 nm in length), with a "string-of-beads" appearance. Longer (approximately 7 nm in width, and >1 microm in length), smoother, and seemingly stiffer fibrils formed on heating aqueous beta-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 beta-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 beta-lactoglobulin, by Raman and infrared spectroscopy confirmed beta-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 beta-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.
Gosal
Walraj S
WS
Department of Life Sciences, King's College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NN, United Kingdom. w.s.gosal@leeds.ac.uk
Clark
Allan H
AH
Ross-Murphy
Simon B
SB
D000682:Amyloid / Q000737:chemistry; D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry*; D002417:Cattle; D005782:Gels; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000737:chemistry; D007477:Ions; D007782:Lactoglobulins / Q000737:chemistry*; D046911:Macromolecular Substances / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D008892:Milk / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012997:Solvents / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D013059:Spectrum Analysis, Raman; D013696:Temperature; D013997:Time Factors; D014867:Water / Q000737:chemistry; D014961:X-Ray Diffraction
D000682:Amyloid; D001672:Biocompatible Materials; D005782:Gels; D007328:Insulin; D007477:Ions; D007782:Lactoglobulins; D046911:Macromolecular Substances; D012997:Solvents; D014867:Water