NMR: PROTEIN PROTEIN INTERACTION & ELECTRON TRANSFER IN CAMPHOR HYDROXYLASE PATH

我们最近提出了一个电子转移模型。 PDX之间的复杂性。和细胞色素P450 cam(细胞色素P450细胞色素P450细胞色素P450 cam) 根据两种蛋白质的结构和诱变结果。。 尽管其他实验室最近的工作提供了实验 对这种模式的支持，它仍然是投机性的，因为我们没有 直接观察到两种蛋白质之间的相互作用 从光谱上看。滴定实验(滴定“N-标记PDX 与细胞色素P101结合，观察‘H’的化学位移变化。 光谱)允许确定PDX的大部分区域 受CYP101存在的影响；然而，它们不提供 关于CYPI01上的铁氧还蛋白结合位点的信息。我们计划 使用同位素标记和多维核磁共振研究来证实 并确定了PDX和CYP101之间的相互作用。我们有 用-90%制备了0.5 mm均一的N‘13C标记的CYPI01样品 氢化氢。初步的‘H，“N-HSQC，HNCA，HN(CO)CA和HNCACB 在600 MHz处获得了光谱，结果是非常好的 很有希望。任务目前正在进行中。

We have recently proposed a model for the electron transfer complex between Pdx. (putidaredoxin) and cytochrome P450cam (CYPI01) based on the structures of both proteins and mutagenesis results. . Despite recent work by other laboratories that provides experimental support for this model, it remains speculative, since we have not directly observed the interactions between the two proteins spectroscopically. Titration experiments (titrating "N-labeled Pdx with CYP101 and observing chemical shift changes in the 'H, "N HSQC spectrum) have allowed determination of the regions of Pdx most affected by the presence of CYP101; however, they provide no information regarding the ferredoxin binding site on CYPI01. We plan to use isotopic labeling with multidimensional NMR studies to confirm and identify the interactions between Pdx and CYP101. We have prepared a 0.5 mM uniformly "N '13C-labeled sample of CYPI01 with -90% deuteration. Preliminary 'H,"N-HSQC, HNCA, HN(CO)CA, and HNCACB spectra have been obtained at 600 MHz and the results are very promising. Assignments are currently underway.