Probing the structure and interactions of crystallin proteins by NMR spectroscopy.
The lens is composed primarily of proteins, the crystallins, at high concentration whose structure and interactions are responsible for lens transparency. As there is no protein turnover in the majority of the lens, crystallin proteins have to be very stable and long-lived proteins. There are three types of crystallin proteins: alpha, beta and gamma, and they all are composed of a variety of subunits. In addition, extensive post-translational modification is undergone by many of the subunits. Determining the structural features and the preferential interactions and associations undergone by the crystallin proteins in the lens is a large and complex experimental undertaking. Some progress has been made in this area by X-ray crystallographic determination of structures for representative examples of the beta- and gamma-crystallins [Slingsby, C., Norledge, B., Simpson, A., Bateman, O. A., Wright, G., Driessen H. P. C., Lindley, P. F., Moss, D. S. and Bax, B. (1997) X-ray diffraction and structure of crystallins. Prog. Ret. Eye Res. 16, 3-29]. In this article, a summary is given of nuclear magnetic resonance (NMR) methods to determine information about these aspects of crystallin proteins. It is shown that despite their relatively large size, all crystallins give rise to well-resolved NMR spectra which arise from flexible terminal extensions that extend from the domain core of the proteins. By examining NMR spectra of mixtures of different crystallin subunits, it is possible to determine the role of these extensions in crystallin-crystallin interactions. For example, the flexible C-terminal extensions in the two alpha-crystallin subunits are not involved in interacting with the other crystallins but are crucially important in the chaperone action of alpha-crystallin. In this action, alpha-crystallin stabilises other proteins under conditions of stress, e.g. heat. In the lens, this ability probably has important consequences in preventing the precipitation of crystallin proteins with age and thereby contributing to cataract formation. The C-terminal extensions in alpha-crystallin act as solubilising agents for the protein and the high-molecular-weight complex that forms upon chaperone action with a precipitating "substrate" protein. Similar behaviour is observed for a variety of small heat-shock proteins, to which alpha-crystallin is related. NMR studies are also consistent with a two-domain structure for alpha-crystallin. No crystal structure is available for crystallin. Using the NMR data, a model for the quaternary structure of alpha-crystallin is proposed which comprises an annular arrangement for the subunits with a large central cavity.
Carver
J A
JA
Department of Chemistry, The University of Wollongong, NSW, Australia. john_carver@uow.edu.au
D000595:Amino Acid Sequence; D000818:Animals; D003459:Crystallins / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D006360:Heat-Shock Proteins / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D016415:Sequence Alignment
D003459:Crystallins; D006360:Heat-Shock Proteins