(a) Amide longitudinal (R_N(N_Z)) and (b) transverse (R_N(N_X)) relaxation rates and (c) ¹⁵N heteronuclear NOE enhancements (hetNOE) are displayed for Pup. Both sets of resonances are displayed for R29 and V55–V59 with the arbitrarily assigned “second” resonance displayed in red. The average value is displayed with a blue line to highlight sequence variations.

(a) CD spectra in the far-UV region are monitored at several temperatures spanning from 20 to 80 °C on Pup by using a spectropolarimeter equipped with a water circulation temperature control. (b) Thermal unfolding transition curves are provided by plotting the change in ellipticity at 197.7 nm from (a) across temperature. No melting transition is observed for Pup.

(a) ¹H, ¹⁵N HSQC titration experiment in which unlabeled Mpa is added to ¹⁵N labeled Pup. Free Pup (black) is displayed superimposed onto Pup:Mpa hexamer at molar ratios of 1:0.08, 1:0.3, and 1:1 as indicated. Resonances from the Mpa-bound state that persist throughout the titration are highlighted with an asterisk, whereas those that appear and then disappear are marked with an arrow. The binding is in slow exchange and therefore the Mpa-bound state is not assigned. (b) Quantitative analysis of the Pup residues involved in Mpa hexamer binding plotting (left panel) or (right panel) for each residue in the Pup sequence. In this equation, I_1:0.3 and I_1:1 is the intensity of the unbound Pup resonance with 0.3 and equimolar ratio of Mpa hexamer; I_free is the intensity of the Pup resonance with no Mpa hexamer present. (c) A model depicting a pupylated substrate binding to Mpa, which forms a hexamer. Pup, its substrate, and Mpa are displayed in orange, blue and grey, respectively.

(a) ¹H, ¹⁵N HSQC experiments reveal that Pup (shown on the left) lacks the amide chemical shift dispersion characteristic of a folded protein (as displayed for ubiquitin on the right). R29, V55–V59 exhibited two sets of amide crosspeaks, as labelled. (b) Chemical shift index (CSI) profile for Pup Cα (left) and Hα (right) atoms are displayed. Residues A51–F54 are highlighted in red, as they demonstrate the shifting characteristic of helical structures and were also demonstrated to be helical in an ¹⁵N dispersed NOESY spectrum (Figure c). CSI values were determined by subtracting random coil chemical shift value from the assigned one. (c) Regions with long-range NOE interactions are displayed for an ¹⁵N dispersed NOESY spectrum acquired on ¹⁵N labeled Pup in 20 mM HEPES (pH 6.5) and 50 mM NaCl; no glycerol was used for this experiment. Intra-residue, sequential, i → i+2, and i → i+3 interactions are displayed in black, red, purple, and blue, respectively. (d) Summary of NOESY and CSI data demonstrating that residues A51–F54 (highlighted in blue) exhibit a propensity towards helicity while the rest of the sequence is largely devoid of canonical secondary structure elements. Residues undergoing slow conformational exchange are indicated in grey.

(a) The amino acid sequence of Pup is displayed with its aspartic and glutamic acids in grey and its diglycine motif and C-terminal glutamine in red and blue, respectively. Its predicted secondary structure (with the program Jpred33) and sequence complexity (with the program seg34) is displayed below the sequence. Helical regions and sequences of low complexity are displayed with ‘H’ and an ‘X,’ respectively. The results of the disorder prediction programs PONDR VSL211 and IUPred12 are plotted as labeled; scores over 0.5 indicate predicted disorder. (b) Size exclusion chromatrography by FPLC reveals that 6.9 kDa Pup has a larger hydrodynamic radius than 8.6 kDa ubiquitin. At the bottom of the chromatogram, we indicate the reported elution volume for ferritin, albumin, ovalbumin and myoglobin with arrows labeled a, b, c, and d, respectively. (c) A ribbon diagram is displayed of ubiquitin to highlight its structural complexity. Ubiquitin targets proteins for degradation in eukaryotes, but unlike Pup forms a well-folded, compact structure. (d) Pup’s apparent R_s based on its elution time in (b) matches that of a natively unfolded protein. Stokes radius, R_s; MW, molecular weight.