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-directed mutagenesis has been used to identify regions of the protein necessary for chaperone function. In this work we have taken some of the previously 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 degrees C. In general the different assays gave parallel results indicating that the same property is being measured. Discrepancies 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 alpha A-crystallin, alpha Ains-crystallin, had decreased chaperone function. Decreased chaperone function was also found for human Ser139--> Arg, Thr144-->Arg, Ser59-->Ala mutants of alpha B-crystallin and double mutants Ser45-->Ala/Ser59-->Ala, Lys103--> Leu/His104-->Ile, and Glu110-->His/His111-->Glu. A mutant Phe27-->Arg that was the subject of previous controversy was shown to be fully active at physiological temperatures.
Derham
B K
BK
Nuffield Laboratory of Ophthalmology, Walton Street, University of Oxford, UK.
van Boekel
M A
MA
Muchowski
P J
PJ
Clark
J I
JI
Horwitz
J
J
Hepburne-Scott
H W
HW
de Jong
W W
WW
Crabbe
M J
MJ
Harding
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003459:Crystallins / Q000737:chemistry* / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009206:Myocardium / Q000201:enzymology; D051381:Rats; D013552:Swine; D013696:Temperature
D003459:Crystallins; D018832:Molecular Chaperones; D008291:Malate Dehydrogenase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases