Sharma K K, Kumar G S, Murphy A S, Kester K
Department of Biochemistry, University of Missouri, Columbia, Missouri 65212, USA.
J Biol Chem. 1998 Jun 19;273(25):15474-8. doi: 10.1074/jbc.273.25.15474.
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.
使用荧光探针1,1'-联（4-苯胺基）萘磺酸（双ANS）、8-苯胺基-1-萘磺酸（ANS）和1-叠氮萘-5-磺酸（1,5-AZNS）评估α-晶状体蛋白中的疏水结合位点。双ANS-α-晶状体蛋白复合物的光解导致探针掺入αA和αB亚基。变性乙醇脱氢酶与α-晶状体蛋白的预先结合显著降低了双ANS对α-晶状体蛋白的光掺入。掺入αA-晶状体蛋白中的双ANS的定位导致鉴定出残基QSLFR和HFSPEDLTVK为荧光团结合区域。在αB-晶状体蛋白中，发现序列DRFSVNLNVK和VLGDVIEVHGK是双ANS结合区域。在双ANS结合序列中，αA-晶状体蛋白的HFSPEDLTVK以及αB-晶状体蛋白的DRFSVNLNVK和VLGDVIEVHGK先前被鉴定为在分子伴侣样作用期间与靶蛋白相互作用的序列的一部分。疏水探针1,5-AZNS也与α-晶状体蛋白的两个亚基相互作用。αB-晶状体蛋白中1,5-AZNS结合位点的定位导致鉴定出HFSPEEK序列为α-晶状体蛋白该亚基中的相互作用位点。糖化的α-晶状体蛋白显示出ANS荧光降低和伴侣样功能丧失，表明糖基化位点以及ANS结合位点参与了伴侣样活性的表现。
