Characterization of Human iNOS Proteins

Expression and purification of the human iNOS proteins were performed as described.^{19 (link),26 (link)} Each protein sample was loaded between two CaF₂ windows separated by a 76 μm spacer. Visible spectra (Agilent Cary 300 Spectrometer) and FT IR spectra (Agilent Cary 670 FT-IR spectrometer) of all samples were recorded to ensure binding of CO. Details regarding the procedure of protein identification by mass spectrometry, Fe(II)–CO sample preparation, FT IR spectral acquisition, and subsequent processing are provided in the Supporting Information. 2D IR spectroscopy was conducted in the traditional BOXCARS geometry as previously described; see the Supporting Information for a complete description.^{27 -29 (link)} The center line slope (CLS) analysis of the T_w-dependent 2D IR spectra along with fitting to the linear FT IR spectra was used to determine FFCFs parameters.^{30 (link),31 (link)} Experiments were performed in triplicate with independently prepared samples, except for the full-length protein, for which we were only able to obtain duplicate data.