Figure 3.

{¹H} -¹⁵N Heteronuclear NOE Data for hN1 Constructs

(A–D) Data are shown for (A) EGF4-7, (B) EGF7-9, (C) EGF8-11 and (D) EGF11-13. Reduced NOE ratios, characteristic of significant mobility on a nanosecond to picosecond timescale, are observed at the N terminus of each construct and for the loop between the first and second Cys in EGF8 and EGF11 (indicated by an asterisk). The regions highlighted by the dashed vertical lines and shading represent the linker between pairs of EGF domains (six residues between the sixth Cys of one domain and the first cysteine of the next for all linkers except EGF9-10 which has five residues). It is clear that reduced NOE ratios are not observed in any of these linkers, indicating that they are not flexible on a fast timescale. EGF4 shows reduced NOE values for residues 158–161. These residues are located in the β turn between the third and fourth Cys; in the X-ray structures of EGF4-7 these residues show high B factors or missing electron density suggesting dynamic behavior. Uncertainties in the NOE ratios were estimated from 500 Monte Carlo simulations using baseline noise as a measure of the error in the peak heights.