PLANT CELL WALL CROSS LINKING VIA HYDROXYCINNAMIC ACIDS

e. 大肠杆菌硫氧还蛋白（Trx）是一种常见的蛋白质二硫键 还原剂 Trx活性位点，位于？-螺旋， 由Cys-Gly-Pro-Cys组成。 该酶在二硫醇和 二硫化物氧化还原态。 第一硫醇（Cys32）具有降低的pKa 相对于典型的蛋白质硫醇。 该pKa可以通过以下方式测量： 观察滴定的C？H和C？使用2QF-COSY的Cys32的H 实验 我们已经产生了Trx突变体， 位点二硫键还原电位。 还原突变体的pH滴定 通过荧光监测的硫氧还蛋白表明， Cys32 这些突变体的二维1H-NMR（2QF-CSY）将允许 Cys32 pKa的准确测定。 物理化学表明， 较低的pKa将与较高的（更正的）还原相关 由于Trx的二硫醇形式的稳定化，

E. coli thioredoxin (Trx) is a general protein disulfide reductant. The Trx active site, at the N-terminus of an ?-helix, consists of Cys-Gly-Pro-Cys. The enzyme converts between dithiol and disulfide redox states. The first thiol (Cys32) has a depressed pKa relative to a typical protein thiol. This pKa can be measured by observing the titration of the C?H and C?H of Cys32 using a 2QF-COSY experiment. We have generated Trx mutants that have perturbed active site disulfide reduction potentials. pH titration of reduced mutant thioredoxins monitored by fluorescence suggest altered pKa's for Cys32. Two-dimensional 1H-NMR (2QF-CSY) of these mutants will allow accurate determination of Cys32 pKa. Physical chemistry suggests that a lower pKa will correlate with a higher (more positive) reduction potential due to stabilization of the dithiol form of Trx.