NADPH-Cytochrome P450 Oxidoreductase: Extraction of the Full-Length Protein and Methyl-TROSY NMR of the Soluble Mutants
Date of Award
Master of Science (MS)
NADPH-cytochrome p450 oxidoreductase (CYPOR) is a membrane-bound protein in living cells. CYPOR delivers electrons to cytochrome p450 proteins (CYPs) to catalyze metabolism of drugs and synthesis of steroids. Extraction and solubilization of CYPOR from the membrane is typically done with the TritonX-100 detergent. The amount of the solubilized protein by this detergent, however, remains relatively low to structurally analyze CYPOR with NMR spectroscopy. The goal of the first project in this thesis was to optimize the amount of the extracted CYPOR from the E. coli membrane using various detergents and additives. To this aim, non-ionic detergents with variable hydrophobicity (TritonX-100, X-114, and X-405) and binding strength to the extracted protein (TritonX-100, TWEEN20, and Brij35) were evaluated. Besides, the combinations of TritonX-100 with CHAPS or polyamine and alkylamine additives were assessed. None of these detergents and additives extracted more of CYPOR than the typical amount extracted by TritonX-100. Thus, it was concluded that this detergent extracts all of the available and functional CYPOR. The remaining protein is probably in an unusual and aggregated form. Understanding the details of CYPOR dynamics can be achieved by solution NMR spectroscopy. The initial step towards this goal requires NMR signal assignments of crucial residues in the protein. In this contribution, NMR analysis was performed on the soluble form of CYPOR lacking its N-terminal hydrophobic anchor (Δ56). Two dual cysteine mutants of this form of the protein (Q157C/Q517C and Q157C/N271C) were reacted with 13C-methyl-methanethiosulfonate (13C-MMTS). The resulting residue, which is 13C -methylthiocysteine (13C-MTC) gave strong signals in the 1H-13C HSQC and 1H-13C HMQC spectra of the mutants. The new assignment of MTC-271 at 2.46 ppm 1H, 25.42 ppm 13C was established besides the existing assignments of MTC-157 and MTC-517. The NMR spectra of the two mutants were highly resolved, and they lacked the middle peak. This peak was previously reported in the 1H-13C HMQC spectra of several Δ56 CYPOR mutants. It was concluded that this unspecific peak is due to sample preparation rather than the NMR technique.