␣-Crystallins are molecular chaperones that protect vertebrate eye lens proteins from detrimental protein aggregation. ␣B-Crystallin, 1 of the 2 ␣-crystallin isoforms, is also associated with myopathies and neuropathological diseases. Despite the importance of ␣-crystallins in protein homeostasis, only little is known about their quaternary structures because of their seemingly polydisperse nature. Here, we analyzed the structures of recombinant ␣-crystallins using biophysical methods. In contrast to previous reports, we show that ␣B-crystallin assembles into defined oligomers consisting of 24 subunits. The 3-dimensional (3D) reconstruction of ␣B-crystallin by electron microscopy reveals a spherelike structure with large openings to the interior of the protein.␣A-Crystallin forms, in addition to complexes of 24 subunits, also smaller oligomers and large clusters consisting of individual oligomers. This propensity might explain the previously reported polydisperse nature of ␣-crystallin.electron microscopy ͉ molecular chaperone ͉ protein aggregation ͉ small heat shock protein ͉ stress response
Self-assembly of beta-sheet domains resulting in the formation of pathogenic, fibrillar protein aggregates (amyloids) is a characteristic feature of various medical disorders. These include neurodegenerative diseases, such as Alzheimer's, Huntington's, and Creutzfeldt-Jacob's. A significant problem in studying such aggregation processes is the poor solubility of these beta-sheet complexes. The present work describes water-soluble de novo beta-sheet peptides which self-assemble into fibrillar structures. The model peptides enable studies of the relationship between beta-sheet stability and association behavior. The peptides [DPKGDPKG-(VT)n-GKGDPKPD-NH2, n = 3-8] are composed of a central beta-sheet-forming domain (VT-sequence), and N- and C-terminal nonstructured octapeptide sequences which promote water solubility. Conformational analyses by circular dichroism and Fourier transform infrared spectroscopy indicate the influence of peptide length, D-amino acid substitution, and concentration on the ability of the peptides to form stable beta-sheet structures. The association behavior investigated by analytical ultracentrifugation and dynamic light scattering was found to correlate strongly with the stability of a beta-sheet conformation. Model peptides with n >/= 6 form stable, water-soluble beta-sheet complexes with molecular masses of more than 2000 kDa, which are organized in fibrillar structures. The fibrils examined by Congo Red staining and electron microscopy show some similarities with naturally occurring amyloid fibrils.
Using dynamic light scattering we show that aqueous super- to undersaturated solutions of NaCl, (NH4)2SO4,
and Na-citrate contain submicrometer size clusters at room temperature. The particle size distributions deduced
by Laplace inversion of the spectra contain two predominant components. The smaller components with radii
below 1 nm are attributed to mixtures of solvated ions and the larger with radii between 50−500 nm to ion
clusters. Knowledge of the mesoscale structure of concentrated electrolyte solutions may be necessary to
describe effective protein−protein interactions which are of importance in biochemical applications such as
protein crystallization.
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