The proposed research will contribute to solving one of the major remaining problems in lens research: the lack of knowledge about the three-dimensional structure nd the biological functions of alpha- crystallin. this does not only seriously hamper a rational approach towards cataract prevention, but is the more urgent considering the involvement of the alphaB-crystallin subunit as a stress-protein in many important pathological conditions outside the lens. Moreover, because of the structural and probably also functional homologies between alpha- crystallin and the small heat-shock proteins, this proposed research is also of direct relevance for a better insight in the properties of this increasingly important family of proteins. The specific objectives of this proposal, exploiting the possibilities of protein engineering techniques, are twofold: 1. Structural - To make site-directed and deletion/insertion mutants of both the alphaA- and alphaB-subunit of human alpha-crystallin in order to produce subunits, and domains thereof, that can be used for quaternary structure studies and that are suitable to obtain detailed tertiary structure information. To achieve this goal separate domains and site- directed mutants of alphaA- and alphaB-crystallin will be produced in E. coli by expression of the desired cDNA constructs. The influence of the mutations on aggregation will be determined. the recombinant proteins will be purified and used for crystallization studies for subsequent X- ray diffraction analysis. In case of monomeric subunits or domains, use will be made of 2D-NMR spectroscopic studies. 2. Functional - to study the effects of engineered mutants and natural variants of alphaA- and alphaB-crystallin on the biological properties of these proteins, such as: aggregation behavior and stability, membrane binding, protease inhibition, chaperone activity, intracellular translocation and thermoprotection, interaction with actin and intermediate filaments, phosphorylation and mRNA binding. In this manner insight of a fundamentally new type can be obtained about the residues and regions in alpha-crystallin that are involved in these various functional aspects. This goal will be approached by subjecting the domains and mutants to a variety of relevant assays. Where possible and relevant the mouse small heat-shock protein HSP25 will be included in the comparison.
