2.4. NMR Spectroscopy

NMR samples were prepared from unlabelled purified peptide (0.2 mm) in 90% H₂O/10% D₂O. All NMR spectra were recorded on a 600 MHz AVANCE III NMR spectrometer (Bruker, Karlsruhe, Germany), equipped with a 5 mm TCI cryoprobe. Two-dimensional ¹H−¹H TOCSY, ¹H−¹H NOESY, ¹H−¹H DQF-COSY and ¹H−¹³C HSQC spectra were acquired at 290 K. Spectra were recorded using an interscan delay of 1 s. NOESY spectra were acquired with mixing times of 200 ms, and TOCSY spectra were acquired with isotropic mixing periods of 80 ms. All spectra were processed using Bruker TopSpin (Version 3.5pl7) and assigned using CCPNMR analysis 2.1, based on the approach described in Wüthrich et al. [25,26]. More than 90% of the protons were assigned and 70% of the Cα and Cβ atoms were unambiguously assigned. Amide temperature coefficients were calculated based on TOCSY spectra directly referenced to DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) and recorded at temperatures ranging from 290 K to 305 K using the method outlined in Cierpicki et al. [27]. Hydrogen bond restraints (Supplementary Table S1) were included in the calculations based on hydrogen bonds identified in preliminary structures and amide protons with temperature coefficients more positive than −4.6 ppb/K.

The αH secondary shifts were determined by subtracting random coil ¹H NMR chemical shifts from the experimental αH chemical shifts [28]. Root mean square deviation (RMSD) values were calculated relative to a mean structure using MOLMOL [29]. The structures and chemical shifts have been deposited into the Protein Data Bank and the Biological Magnetic Resonance Data Bank (ZF-N_{24_3}—PDB ID: 7JIA, BMRB ID: 30780; ZF-para_{_3s}—PDB ID: 7JIY, BMRB ID: 30781).