NMR-SPECTROSCOPY OF ALPHA-CRYSTALLIN - INSIGHTS INTO THE STRUCTURE, INTERACTIONS AND CHAPERONE ACTION OF SMALL HEAT-SHOCK PROTEINS

The subunit molecular mass of alpha-crystallin, like many small heat-s hock proteins (sHsps), is around 20 kDa although the protein exists as a large aggregate of average mass around 800 kDa. Despite this large size, a well-resolved H-1 NMR spectrum is observed for alpha-crystalli n which arises from short, polar, highly-flexible and solvent-exposed C-terminal extensions in each of the subunits, alpha A- and alpha B-cr ystallin. These extensions are not involved in interactions with other proteins (e.g. beta- and gamma-crystallins) under non-chaperone condi tions. As determined by NMR studies on mutants of alpha A-crystallin w ith alterations in its C-terminal extension, the extensions have an im portant role in acting as solubilising agents for the relatively-hydro phobic alpha-crystallin molecule and the high-molecular-weight (HMW) c omplex that forms during the chaperone action. The related sHsp, Hsp25 , also exhibits a flexible C-terminal extension. Under chaperone condi tions, and in the HMW complex isolated from old lenses, the C-terminal extension of the alpha A-crystallin subunit maintains its flexibility whereas the alpha B-crystallin subunit loses, at least partially, its flexibility, implying that it is involved in interaction with the 'su bstrate' protein. The conformation of 'substrate' proteins when they i nteract with alpha-crystallin has been probed by H-1 NMR spectroscopy and it is concluded that alpha-crystallin interacts with 'substrate' p roteins that are in a disordered molten globule state, but only when t his state is on its way to large-scale aggregation and precipitation. By monitoring the H-1 and P-31 NMR spectra of alpha-crystallin in the presence of increasing concentations of urea, it is proposed that alph a-crystallin adopts a two-domain structure with the larger C-terminal domain unfolding first in the presence of denaturant. All these data h ave been combined into a model for the quaternary structure of alpha-c rystallin. The model has two layers each of approximately 40 subunits arranged in an annulus or toroid. A large central cavity is present wh ose entrance is ringed by the flexible C-terminal extensions. A large hydrophobic region in the aggregate is exposed to solution and is avai lable for interaction with 'substrate' proteins during the chaperone a ction. (C) 1998 Elsevier Science B.V. All rights reserved.