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

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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.