Abstract
Small heat shock proteins function in a chaperone-like manner to prevent the precipitation of proteins under conditions of stress (e. g. heat). alpha-Crystallin, the major mammalian lens protein, is a small heat shock protein. The mechanism of chaperone action of these proteins is poorly understood. In this paper, the conformational state of a protein when it forms a high molecular weight complex with alpha-crystallin is investigated by examining, using NMR spectroscopy and size exclusion high performance liquid chromatography, the interaction of alpha-crystallin with alpha-lactalbumin and its various intermediately folded (molten globule) states. The complex is formed following reduction of alpha-lactalbumin by dithiothreitol in the presence of alpha-crystallin, and this interaction has been monitored in real time by 1H NMR spectroscopy. It is concluded that alpha-crystallin interacts with a disordered molten globule state of alpha-lactalbumin while it is on an irreversible pathway toward aggregation and precipitation. alpha-Crystallin does not interact, however, with molten globule states of alpha-lactalbumin that are stable in solution, e.g. the reduced and carboxyamidated species. It is proposed that alpha-crystallin distinguishes between the various molten globule states of alpha-lactalbumin on the basis of the lifetimes of these states, i.e. the protein must be in a disordered molten globule state for a significant length of time and on the pathway to aggregation and precipitation for interaction to occur.
Highlights
It has become apparent over the last few years that the folding of proteins in vivo involves the interaction of nascent polypeptides with a diversity of unrelated proteins known as chaperones (reviewed by Buchner (1))
Chaperone proteins are often known as heat shock proteins (Hsps) because of their increased expression when cells are exposed to elevated temperatures
Under similar conditions to those for insulin, ␣-crystallin prevents the precipitation of apo␣-lactalbumin from solution (Fig. 1)
Summary
It has become apparent over the last few years that the folding of proteins in vivo involves the interaction of nascent polypeptides with a diversity of unrelated proteins known as chaperones (reviewed by Buchner (1)). From the results presented, ␣crystallin stabilizes ␣-lactalbumin in the A state, and because no further changes are observed with time in the NMR spectrum of ␣-lactalbumin in the presence of ␣-crystallin (apart from a decrease in intensity of these broadened ␣-lactalbumin resonances), this disordered molten globule state interacts with ␣-crystallin.
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