Maeda Kenji, Finnie Christine, Svensson Birte
Department of Chemistry, Carlsberg Laboratory, Copenhagen, Denmark.
Proteomics. 2005 Apr;5(6):1634-44. doi: 10.1002/pmic.200401050.
Using thiol-specific fluorescence labelling, over 30 putative target proteins of thioredoxin h with diverse structures and functions have been identified in seeds of barley and other plants. To gain insight at the structural level into the specificity of target protein reduction by thioredoxin h, thioredoxin h-reducible disulphide bonds in individual target proteins are identified using a novel strategy based on differential alkylation of cysteine thiol groups by iodoacetamide and 4-vinylpyridine. This method enables the accessible cysteine side chains in the thiol form (carbamidomethylated) to be distinguished from those inaccessible or disulphide bound form (pyridylethylated) according to the mass difference in the peptide mass maps obtained by matrix-assistend laser desorption/ionisation-time of flight mass spectrometry. Using this approach, in vitro reduction of disulphides in recombinant barley alpha-amylase/subtilisin inhibitor (BASI) by barley thioredoxin h isoform 1 was analysed. Furthermore, the method was coupled with two-dimensional electrophoresis for convenient thioredoxin h-reducible disulphide identification in barley seed extracts without the need for protein purification or production of recombinant proteins. Mass shifts of 15 peptides, induced by treatment with thioredoxin h and differential alkylation, identified specific reduction of nine disulphides in BASI, four alpha-amylase/trypsin inhibitors and a protein of unknown function. Two specific disulphides, located structurally close to the alpha-amylase binding surfaces of BASI and alpha-amylase inhibitor BMAI-1 were demonstrated to be reduced to a particularly high extent. For the first time, specificity of thioredoxin h for particular disulphide bonds is demonstrated, providing a basis to study structural aspects of the recognition mechanism and regulation of target proteins.
利用硫醇特异性荧光标记，在大麦和其他植物种子中已鉴定出30多种结构和功能各异的硫氧还蛋白h的假定靶蛋白。为了在结构水平上深入了解硫氧还蛋白h对靶蛋白还原的特异性，采用一种基于碘乙酰胺和4-乙烯基吡啶对半胱氨酸硫醇基团进行差异烷基化的新策略，来鉴定单个靶蛋白中可被硫氧还蛋白h还原的二硫键。该方法能够根据基质辅助激光解吸/电离飞行时间质谱获得的肽质量图谱中的质量差异，区分处于硫醇形式（氨基甲酰甲基化）的可及半胱氨酸侧链与那些不可及或处于二硫键结合形式（吡啶基乙基化）的侧链。采用这种方法，分析了大麦硫氧还蛋白h同工型1对重组大麦α-淀粉酶/枯草杆菌蛋白酶抑制剂（BASI）中二硫键的体外还原作用。此外，该方法与二维电泳相结合，便于在大麦种子提取物中鉴定可被硫氧还蛋白h还原的二硫键，而无需进行蛋白质纯化或生产重组蛋白。用硫氧还蛋白h处理并进行差异烷基化后，15个肽段的质量发生了变化，这确定了BASI、4种α-淀粉酶/胰蛋白酶抑制剂和一种功能未知的蛋白中9个二硫键的特异性还原。已证明，在结构上靠近BASI和α-淀粉酶抑制剂BMAI-1的α-淀粉酶结合表面的两个特定二硫键被还原的程度特别高。首次证明了硫氧还蛋白h对特定二硫键的特异性，为研究靶蛋白识别机制和调控的结构方面提供了基础。
