Identification of a region in alcohol dehydrogenase that binds to alpha-crystallin during chaperone action.
alpha-Crystallin, the major eye lens protein and a member of the small heat-shock protein family, has been shown to protect the aggregation of several proteins and enzymes under denaturing conditions. The region(s) in the denaturing proteins that interact with alpha-crystallin during chaperone action has not been identified. Determination of these sites would explain the wide chaperoning action (promiscuity) of alpha-crystallin. In the present study, using two different methods, we have identified a sequence in yeast alcohol dehydrogenase (ADH) that binds to alpha-crystallin during chaperone-like action. The first method involved the incubation of alpha-crystallin with ADH peptides at 48 degrees C for 1 h followed by separation and analysis of bound peptides. In the second method, alpha-crystallin was first derivatized with a photoactive trifunctional cross-linker, sulfosuccinimidyl-2[6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]ethyl-1,3di-thiopropionate (sulfo-SBED), and then complexed with ADH at 48 degrees C for 1 h in the dark. The complex was photolyzed and digested with protease, and the biotinylated peptide fragments were isolated using an avidin column and then analyzed. The amino acid sequencing and mass spectral analysis revealed the sequence YSGVCHTDLHAWHGDWPLPVK (yeast ADH(40-60)) as the alpha-crystallin binding site in ADH. The interaction was further confirmed by demonstrating complex formation between alpha-crystallin and a synthetic peptide representing the binding site of ADH.
Santhoshkumar
Puttur
P
Department of Ophthalmology, University of Missouri, Columbia, MO 65212, USA.
Sharma
Krishna K
KK
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003459:Crystallins / Q000737:chemistry / Q000378:metabolism*; D007908:Lens, Crystalline / Q000737:chemistry; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D003459:Crystallins; D018832:Molecular Chaperones; D010446:Peptide Fragments; D000426:Alcohol Dehydrogenase