Chaperone function of mutant versions of alpha A- and alpha B-crystallin prepared to pinpoint chaperone binding sites

A major stress protein, alpha -crystallin, functions as a chaperone. Site-d irected mutagenesis has been used to identify regions of the protein necess ary for chaperone function. In this work we have taken some of the previous ly described mutants produced and assessed their chaperone function by both a traditional heat-induced aggregation method at elevated temperature and using enzyme methods at 37 degreesC. In general the different assays gave p arallel results indicating that the same property is being measured. Discre pancies were explicable by the heat lability of some mutants. Most mutants had full chaperone function showing the robust nature of alpha -crystallin. A mutant corresponding to a minor component of rodent alphaA-crystallin, a lpha Ains-crystallin, had decreased chaperone function. Decreased chaperone function was also found for human Ser139--> Arg, Thr144-->Arg, Ser59-->Ala mutants of alphaB-crystallin and double mutants Ser45-->Ala/Ser59-->Ala, L ys103--> Leu/His104-->Ile, and Glu110-->His/His111-->Glu. A mutant Phe27--> Arg that was the subject of previous controversy was shown to be fully acti ve at physiological temperatures.