Probing the structure and interactions of crystallin proteins by NMR spectroscopy

The lens is composed primarily of proteins, the crystallins, at high concen tration whose structure and interactions are responsible for lens transpare ncy. As there is no protein turnover in the majority of the lens, crystalli n 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 compo sed of a variety of subunits. In addition, extensive post-translational mod ification is undergone by many of the subunits. Determining the structural features and the preferential interactions and associations undergone by th e crystallin proteins in the lens is a large and complex experimental under taking. Some progress has been made in this area by X-ray crystallographic determination of structures for representative examples of the beta- and ga mma-crystallins [Slingsby, C., Norledge, B., Simpson, A., Bateman, O. A., W right, G., Driessen H. P. C., Lindley, P. F., Moss, D. S. and Bar. 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 (NM R) methods to determine information about these aspects of crystallin prote ins. It is shown that despite their relatively large size, all crystallins give rise to well-resolved NMR spectra which arise from flexible terminal e xtensions that extend from the domain core of the proteins. By examining NM R spectra of mixtures of different crystallin subunits, it is possible to d etermine the role of these extensions in crystallin-crystallin interactions . For example, the flexible C-terminal extensions in the two alpha-crystall in subunits are not involved in interacting with the other crystallins but are crucially important in the chaperone action of alpha-crystallin. In thi s action, alpha-crystallin stabilises other proteins under conditions of st ress, e.g. heat. In the lens, this ability probably has important consequen ces in preventing the precipitation of crystallin proteins with age and the reby contributing to cataract formation. The C-terminal extensions in alpha -crystallin act as solubilising agents for the protein and the high-molecul ar-weight complex that forms upon chaperone action with a precipitating "su bstrate" 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 st ructure is available for alpha-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. (C) 1999 Elsevier Science Ltd. All rights reserved.