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Alpha-crystallin is the most important soluble protein in the eye lens. It is responsible for creating a high refractive index and is known to be a small heat-shock protein. We have used static and dynamic light scattering to study its quaternary structure as a function of isolation conditions, temperature, time, and concentration. We have used tryptophan fluorescence to study the temperature dependence of the tertiary structure and its reversibility. Gel filtration, analytical ultracentrifugation, polyacrylamide gel electrophoretic analysis, and absorption measurements were used to study the chaperone-like activity of alpha-crystallin in the presence of destabilized lysozyme. We have demonstrated that the molecular mass of the in vivo alpha-crystallin oligomer is about 700 kDa (alpha(native)) while the 550 kDa molecule (alpha(37 degrees C),diluted), which is often found in vitro, is a product of prolonged storage at 37 degrees C of low concentrated alpha-crystallin solutions. We have proven that the molecular mass of the alpha-crystallin oligomer is concentration dependent at 37 degrees C. We have found strong indications that, during chaperoning, the alpha-crystallin oligomer undergoes a drastic rearrangement of its peptides during the process of complex formation with destabilized lysozyme. We propose the hypothesis that all these processes are governed by the phenomenon of subunit exchange, which is well-known to be strongly temperature-dependent.

We have studied the interaction between lysozyme, destabilized by reducing its -S-S- bonds, and bovine eye lens alpha-crystallin, a member of the alpha-small heat shock protein superfamily. We have used gel filtration, photon correlation spectroscopy, and analytical ultracentrifugation to study the binding of lysozyme by alpha-crystallin at 25 degrees C and 37 degrees C. We can conclude that alpha-crystallin chaperones the destabilized protein in a two-step process. First the destabilized proteins are bound by the alpha-crystallin so that nonspecific aggregation of the destabilized protein is prevented. This complex is unstable, and a reorganization and inter-particle exchange of the peptides result in stable and soluble large particles. alpha-Crystallin does not require activation by temperature for the first step of its chaperone activity as it prevents the formation of nonspecific aggregates at 25 degrees C as well as at 37 degrees C. The reorganization of the peptides, however, gives rise to smaller particles at 37 degrees C than at 25 degrees C. Indirect evidence shows that the association of several alpha-crystallin/substrate protein complexes leads to the formation of very large particles. These are responsible for the increase of the light scattering.

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Lens aA-and aB-crystallin have been reported to act differently in their protection against nonthermal destabilization of proteins. The nature of this difference, however, is not completely understood. Therefore we used a combination of thermally and solvent-induced structural changes to investigate the difference in the secondary, tertiary and quaternary structures of aA-and aB-crystallin. We demonstrate the relationship between the changes in the tertiary and quaternary structures for both polypeptides. Far-ultraviolet circular dichroism revealed that the secondary structure of aB-crystallin is more stable than that of aA-crystallin, and the temperature-induced secondary structure changes of both polypeptides are partially reversible. Tryptophan fluorescence revealed two distinct transitions for both aA-and aB-crystallin. Compared to aB-crystallin, both transitions of aA-crystallin occurred at higher temperature. The changes in the hydrophobicity are accompanied by changes in the quaternary structure and are biphasic, as shown by bis-1-anilino-8-naphthalenesulfonate fluorescence and sedimentation velocity. These phenomena explain the difference in the chaperone capacity of aA-and aB-crystallin carried out at different temperatures. The quaternary structure of a-crystallin is more stable than that of aA-and aB-crystallin. The latter has a strong tendency to dissociate under thermal or solvent destabilization. This phenomenon is related to the difference in subunit organization of aA-and aB-crystallin where both hydrophobic and ionic interactions are involved. We find that an important subunit rearrangement of aA-crystallin takes place once the molecule is destabilized. This subunit rearrangement is a requisite phenomenon for maintaining a-crystallin in its globular form and as a stable complex. On the base of our results, we suggest a four-state model describing the folding and dissociation of aA-and aB-crystallin better than a three-state model [Sun et al. (1999) Keywords: analytical ultracentrifugation; circular dichroism; fluorescence; aA-crystallin; aB-crystallin.Small heat-shock proteins (sHSPs) are molecular chaperones that can interact with other proteins to prevent their heatinduced aggregation [2]. The most widely studied sHSPs are the diverse proteins with small molecular masses in the range 15 to 30 kDa. These proteins are known to form large particles in the 300±800 kDa range for mammalian groups [3], and from 200 to 240 kDa for the plant sHSPs [4]. a-Crystallin is the largest soluble protein in the mammalian eye lens cytoplasm and its concentration is by far the highest. Its broad distribution of sizes can range from 300 kDa to 1.5 MDa, depending on the isolation conditions (temperature, solvent and concentration) [5] or the age of the tissue from which the protein is isolated. It is considered to play a structural role in maintaining the refractive properties and the transparency of the lens [6]. a-Crystallin, a member of the sHSPs, has been shown to play an important role in suppressing the heat-...