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The molecular chaperone, alpha-crystallin, has the ability to prevent the fibrillar aggregation of proteins implicated in human diseases, for example, amyloid beta peptide and alpha-synuclein. In this study, we examine, in detail, two aspects of alpha-crystallin's fibril-suppressing ability: (a) its temperature dependence, and (b) the nature of the aggregating species with which it interacts. First, the efficiency of alpha-crystallin to suppress fibril formation in kappa-casein and alpha-synuclein increases with temperature, despite their rate of fibrillation also increasing in the absence of alpha-crystallin. This is consistent with an increased chaperone ability of alpha-crystallin at higher temperatures to protect target proteins from amorphous aggregation [GB Reddy, KP Das, JM PetrashWK Surewicz (2000) J Biol Chem275, 4565-4570]. Second, dual polarization interferometry was used to monitor real-time alpha-synuclein aggregation in the presence and absence of alphaB-crystallin. In contrast to more common methods for monitoring the time-dependent formation of amyloid fibrils (e.g. the binding of dyes like thioflavin T), dual polarization interferometry data did not reveal any initial lag phase, generally attributed to the formation of prefibrillar aggregates. It was shown that alphaB-crystallin interrupted alpha-synuclein aggregation at its earliest stages, most likely by binding to partially folded monomers and thereby preventing their aggregation into fibrillar structures.

alpha-Crystallin is the principal lens protein which, in addition to its structural role, also acts as a molecular chaperone, to prevent aggregation and precipitation of other lens proteins. One of its two subunits, alphaB-crystallin, is also expressed in many nonlenticular tissues, and a natural missense mutation, R120G, has been associated with cataract and desmin-related myopathy, a disorder of skeletal muscles [Vicart P, Caron A, Guicheney P, Li Z, Prevost MC, Faure A, Chateau D, Chapon F, Tome F, Dupret JM, Paulin D & Fardeau M (1998) Nat Genet20, 92-95]. In the present study, real-time 1H-NMR spectroscopy showed that the ability of R120G alphaB-crystallin to stabilize the partially folded, molten globule state of alpha-lactalbumin was significantly reduced in comparison with wild-type alphaB-crystallin. The mutant showed enhanced interaction with, and promoted unfolding of, reduced alpha-lactalbumin, but showed limited chaperone activity for other target proteins. Using NMR spectroscopy, gel electrophoresis, and MS, we observed that, unlike the wild-type protein, R120G alphaB-crystallin is intrinsically unstable in solution, with unfolding of the protein over time leading to aggregation and progressive truncation from the C-terminus. Light scattering, MS, and size-exclusion chromatography data indicated that R120G alphaB-crystallin exists as a larger oligomer than wild-type alphaB-crystallin, and its size increases with time. It is likely that removal of the positive charge from R120 of alphaB-crystallin causes partial unfolding, increased exposure of hydrophobic regions, and enhances its susceptibility to proteolysis, thus reducing its solubility and promoting its aggregation and complexation with other proteins. These characteristics may explain the involvement of R120G alphaB-crystallin with human disease states.