Identification of 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid binding sequences in alpha-crystallin.
The hydrophobic binding sites in alpha-crystallin were evaluated using fluorescent probes 1,1'-bi(4-anilino)naphthalenesulfonic acid (bis-ANS), 8-anilino-1-naphthalene sulfonate (ANS), and 1-azidonaphthalene 5-sulfonate (1,5-AZNS). The photolysis of bis-ANS-alpha-crystallin complex resulted in incorporation of the probe to both alphaA- and alphaB-subunits. Prior binding of denatured alcohol dehydrogenase to alpha-crystallin significantly decreased the photoincorporation of bis-ANS to alpha-crystallin. Localization of bis-ANS incorporated into alphaA-crystallin resulted in the identification of residues QSLFR and HFSPEDLTVK as the fluorophore binding regions. In alphaB-crystallin, sequences DRFSVNLNVK and VLGDVIEVHGK were found to be the bis-ANS binding regions. Of the bis-ANS binding sequences, HFSPEDLTVK of alphaA-crystallin and DRFSVNLNVK and VLGDVIEVHGK of alphaB-crystallin were earlier identified as part of the sequences involved in their interaction with target proteins during the molecular chaperone-like action. The hydrophobic probe, 1,5-AZNS, also interacted with both subunits of alpha-crystallin. Localization of 1,5-AZNS binding site in alphaB-crystallin lead to the identification of HFSPEEK sequence as the interacting site in this subunit of alpha-crystallin. Glycated alpha-crystallin displayed decreased ANS fluorescence and loss of chaperone-like function, suggesting the involvement of glycation site as well as ANS binding site in chaperone-like activity display.
Sharma
K K
KK
Department of Biochemistry, University of Missouri, Columbia, Missouri 65212, USA.
Kumar
G S
GS
Murphy
A S
AS
Kester
K
K
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism*; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000378:metabolism; D003459:Crystallins / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D006031:Glycosylation; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010777:Photochemistry; D011489:Protein Denaturation
D000817:Anilino Naphthalenesulfonates; D003432:Cross-Linking Reagents; D003459:Crystallins; D005456:Fluorescent Dyes; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate)