Crystallins are the principal structural proteins of the eye lens, comprising 90% of the soluble protein. Conservation of sequence within the yield- and BetaGamma-crystallin families suggest that stringent requirements exist for the structure of these proteins for maintenance of lens transparency. This project will examine the relationship of crystallin structure to the properties of protein stability and intermolecular associations and the effect changes in these properties produce on lens transparency. Objectives of this project include delineation of structure-function relationships for the lens crystallins and investigation into the role of crystallins in cataractogenesis. The yield-crystallin promoter will be ligated to the 5' end of modified yield- and Beta-crystallin sequences. These constructions will be used to create transgenic mice expressing abnormal amounts or types of crystallin proteins. One construction will contain the yieldA- or Beta23-crystallin coding regions in an inverted orientation. This should produce an antisense RNA which will hybridize with the natural mRNAs in the cell and inhibit their expression as proteins. Modifications of the Beta-crystallin coding sequence will take advantage of the large amount of structural and sequence information available about this family of proteins. Specific amino acids felt to be vital for maintaining the Greek key motif structures will be altered by site specific mutagenesis. The N-terminal arms, felt to be important in intermolecular associations, will be modified, deleted, or interchanged. The expression and effects of these constructions on crystallin structure and interactions will be monitored by clinical ophthalmological examination, Southern blot analysis (for DNA), Northern blot and S1 analysis (for RNA) and a combination of gel electrophoresis, sieve chromatography, immunoprecipitation and membrane binding analysis at the protein level. These studies will provide insight into the importance of crystallin structure for the functions of stability and protein interactions as well as delineating the role of lens crystallins in hereditary cataracts.
