FIG. 2.

Identification of nonprotein amide resonances. (A) Expansion of the ¹H-¹⁵N HSQC spectrum of HIFd with copurified ECA (black) and HPLC purified ECA (red). Signals from the amides of the N-acetyl groups of ECA are characterized by notably narrow line widths and signal doubling compared to protein signals. (B) CBCA(CO)NH and HNCACB strips for each N-acetyl group of ECA. Black and red indicate positive and negative cross peaks, respectively. These spectra correlate carbon resonances with the amide ¹⁵N and ¹H shifts. In particular, the CBCA(CO)NH experiment gives a positive cross peak between the C_α-like site to the carbonyl (C_α[H₃]). The HNCACB experiment shows positive cross peaks for the same C_α-like site (C_α[H₃]) and the C_α-like site with respect to the HN (C_α) with the same amide. In addition, negative cross peaks are observed in the HNCACB experiment for the C_β-like signals (see header). Note that the C_β-like anomeric signals (≈100 ppm) occur outside of the chemical shift range of the ¹³C dimension (15 to 75 ppm) and are aliased into this range near 40 ppm. Arrows indicate the characteristic signal doubling of ECA_CYC signals, the origin of which may be sample heterogeneity or slow time scale dynamics (see Discussion).