AlphaB-crystallin is a versatile protein. It is one of the elements essential to maintaining the transparency of the eye-lens. AlphaB-crystallin is also found up-regulated in a variety of neuro-degenerative diseases such as Alzheimer's disease and Alexander's disease. Recently, alphaB-crystallin has been shown to be a novel regulator of apoptosis. AlphaB-crystallin has been shown in vitro to be a molecular chaperone and is now classified as belonging to the family of small heat shock proteins (sHSPs) that are induced under conditions of stress. However, alphaB-crystallin (~650 kDa, ~32 subunits, 175 residues per subunit) has remained refractive to high-resolution structure studies like X-ray crystallography and solution-state NMR using conventional methods due to its the dynamic, oligomeric nature. Recent revolutionary technological and methodological advances in NMR will be used to study this challenging system using a """"""""divide and conquer"""""""" approach. A 100-residue core domain from human alphaB forms a dimer under certain conditions of pH and temperature. The structure of the dimeric form (22.8 kDa) of the core domain will be determined using high-resolution, solution state NMR. Agents such as sodium deoxycholate are likely to dissociate the large alphaB complex into tetrameric units, analogous to acrystallin. Detailed solution state NMR studies will be performed on the alphaB-tetramer to determine the structure and orientation of the termini (which are missing in the dimeric core domain ) and thus construct a picture of """"""""mini"""""""" alphaB. Finally, attempts will be made to obtain NMR spectra of the entire oligomeric form of alphaB using recent innovations in NMR. To understand the mechanism of chaperone-substrate interaction, NMR spectra of the core domain plus substrate will acquired using substrates like gamma-crystallin. The effects of ATP will also be determined by comparing spectra of ATP-free and ATP-bound (i) core domain and (ii) `B. Mutants and a chimeric version of alphaB-crystallin will be made to understand the functional aspects of this molecule. To supplement the information obtained from NMR, limited proteolysis mapping will be performed on the core domain/substrate and alphaB/substrate complexes to identify regions of protection. These studies will lay the foundation for more detailed investigations in the future regarding sequence determinants in the various biological roles of alphaB. If attempts to obtain spectra of the 650 kDa molecule are successful, it will open up vast avenues in NMR for studying macromolecules and their function.
