Functional elements in molecular chaperone alpha-crystallin: identification of binding sites in alpha B-crystallin.
alpha-Crystallin, the predominant eye lens protein with sequence homology to small heat shock proteins, acts like a molecular chaperone by suppressing the aggregation of damaged crystallins and proteins. To gain an insight into the amino acid sequences in alpha-crystallin involved in chaperone-like function, we used a cleavable, fluorescent, photoactive, crosslinking agent, sulfosuccinimidyl-2 (7-azido-4-methylcoumarin-3-acetamido)-ethyl-1,3' dithiopropionate (SAED), to derivatize yeast alcohol dehydrogenase (ADH) and allowed it to complex with bovine alpha-crystallin at 48 degrees C. The complex was photolyzed and reduced with DTT and the subunits of alpha-crystallin, alpha A- and alpha B-, were separated. Fluorescence analysis showed that both alpha A- and alpha B-crystallins interacted with ADH during chaperone-like function. Tryptic digestion, amino acid sequencing, and mass spectral analysis of alpha B-crystallin revealed that APSWIDTGLSEMR (57-69) and VLGDVIEVHGKHEER (93-107) sequences were involved in binding with ADH.
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Mason Eye Institute, Department of Ophthalmology, University of Missouri, Columbia 65212, USA. opthks@showme.missouri.edu
Kaur
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Kester
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003459:Crystallins / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D008027:Light; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003459:Crystallins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones