Pierce Anson, deWaal Eric, Van Remmen Holly, Richardson Arlan, Chaudhuri Asish
Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, USA.
Biochemistry. 2006 Mar 7;45(9):3077-85. doi: 10.1021/bi052031i.
Changes in surface hydrophobicity are generally considered as a sensitive indicator for monitoring the structural alterations of proteins that are often associated with changes in function. Currently, no technique has been developed to screen a complex mixture of proteins for changes in the conformation of specific proteins. In this study, we adapted a UV photolabeling approach, using an apolar fluorescent probe, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (BisANS), to monitor changes in surface hydrophobic domains in either purified rhodanese or skeletal muscle cytosolic proteins by urea-induced unfolding or in response to in vitro metal-catalyzed oxidation. Using two-dimensional polyacrylamide gel electrophoresis (2D PAGE), we identified two specific proteins in skeletal muscle cytosol that exhibited a marked loss of incorporation of BisANS after exposure to in vitro oxidative stress: creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found that the activities of both enzymes were also reduced significantly in response to oxidative stress. We then determined if this method could detect changes in surface hydrophobicity in specific proteins arising from oxidative stress generated in vivo by muscle denervation. A loss in surface hydrophobic domains in CK and GAPDH was again observed as measured by the BisANS photoincorporation approach. In addition, the CK and GAPDH activity in denervated muscle was markedly reduced. These data demonstrate for the first time that this assay can screen a complex mixture of proteins for alterations in surface hydrophobic domains of individual proteins.
表面疏水性的变化通常被视为监测蛋白质结构改变的敏感指标，而蛋白质结构改变往往与功能变化相关。目前，尚未开发出能够筛选复杂蛋白质混合物中特定蛋白质构象变化的技术。在本研究中，我们采用了一种紫外线光标记方法，使用非极性荧光探针4,4'-二苯胺基-1,1'-联萘-5,5'-二磺酸（BisANS），通过尿素诱导的去折叠或响应体外金属催化氧化，来监测纯化的硫氰酸酶或骨骼肌胞质蛋白表面疏水结构域的变化。利用二维聚丙烯酰胺凝胶电泳（2D PAGE），我们在骨骼肌胞质中鉴定出两种特定蛋白质，它们在暴露于体外氧化应激后，BisANS的掺入量显著减少：肌酸激酶（CK）和甘油醛-3-磷酸脱氢酶（GAPDH）。我们发现，这两种酶的活性在氧化应激反应中也显著降低。然后，我们确定该方法是否能够检测由肌肉去神经支配在体内产生的氧化应激所导致的特定蛋白质表面疏水性变化。通过BisANS光掺入法测量，再次观察到CK和GAPDH表面疏水结构域的丧失。此外，去神经支配肌肉中的CK和GAPDH活性显著降低。这些数据首次证明，该检测方法能够筛选复杂蛋白质混合物中单个蛋白质表面疏水结构域的改变。
