Figure 1.

Isolated Nsp8 NTD adopts two central α-helices bordered by flexible termini. (A) 2D ¹H–¹⁵N HSQC spectrum of Nsp8 NTD recorded at 5.0°C, in 50 mM NaCl, 10 mM KH₂PO₄, pH 6.1. Assigned backbone ¹H–¹⁵N resonances are labeled. A set of sharper, more intense peaks with less ¹H dispersion corresponding to N- and C-terminal residues are found amid a larger set of resonances with the wide range of ¹HN chemical shifts characteristic of well folded proteins. (B) ¹³Cα (blue bars) conformational chemical shifts (Δδ) reveal two highly populated α-helical structures spanning residues 11–27 and 32–50 (gray shading), as judged by the Δδ ¹³Cα value expected for 100% α-helix. Following residue 50, the helix continues, but its population declines in intensity (light gray shading). The thin line at 3.1 ppm marks the average Δδ¹³Cα value seen for fully populated α-helices. (C) Superposition of the family of 20 conformers of the Nsp8 NTD monomer with side chains colored black and mainchains colored blue. No side chains are shown for the terminal disordered zones. (D) The heteronuclear {¹H}–¹⁵N NOE. High ratios approaching the value of 0.86 (thin line) expected for full rigidity on ns/ps timescales are observed for the two main helical regions. (E) Longitudinal (R₁) and (F) transverse (R₂) relaxation rates gauge μs/ms timescale dynamics. Their relatively low (R₁) and high (R₂) values reflect stiffness.