Interactions with the b5 domain induce helical structure in the N-terminal region.
To compare intrinsic structural properties of the N-terminal region of human Ncb5or with those it exhibits in the native protein, we performed CD spectroscopy studies of the 50-residue N-terminal polypeptide (hereafter N-term) and protein constructs representing the b5 domain with and without the N-terminal region attached (hereafter N/b5 and b5, respectively). Far-UV CD spectra of b5 and N/b5 are shown inFigure 2A (summarized in Table 1) and presented in molar ellipticity units to better reflect differences in total secondary structure content. The N/b5 spectrum differs from that of b5 in exhibiting enhanced negative ellipticity in the vicinity of 200 nm. The dominant feature in far-UV CD spectra of unstructured (random coil) polypeptides is a broad band of negative ellipticity centered near 200 nm.37 The data in Figure 2A therefore suggests that N/b5 differs from b5 in having a larger number of disordered residues. This was confirmed by subtracting the b5 spectrum from the N/b5 spectrum to give the difference spectrum shown in Figure 2C . However, this difference spectrum also exhibits positive ellipticity near 190 nm and negative ellipticity in the vicinity of 220 nm, both suggestive of the presence of helical structure.38 In contrast, the corresponding N-term spectrum (Figure 2C ) is dominated by a negative band centered at 200 nm, revealing it to be largely unstructured. These results strongly suggest that the N-terminal region of Ncb5or is intrinsically disordered, consistent with the predictions described above, but that it adopts some helical structure when connected to the b5 domain.