*4.3. Near-Ultraviolet Circular Dichroism Spectroscopy*

The NUV-CD spectra were obtained on an Applied Photophysics qCD Chirascan spectropolarimeter at ambient temperature. The protein samples were analyzed at a concentration of about 0.5 mg/mL (both folded and unfolded). Using cuvettes with a pathlength of 1 cm, the spectra were corrected for concentration and contributions from the buffer and are reported as Mean Residue Molar Ellipticity. Each spectrum is an average of 4 scans and was smoothed with a 7-point smoothing function using the OMNIC 32 software (Thermo Fisher Scientific Inc.). Background nitrogen blanks and buffer blanks were measured to eliminate the signals from the nitrogen, cuvette, and buffer. The parameters for the Near UV CD were: 1 cm path length, 240–350 nm wave range, 2 s exposure time, 1 nm bandwidth, 0.5 nm step, and averaged over 4 runs with a 900 μL sample volume.

#### *4.4. Nuclear Magnetic Resonance*

A Bruker Avance III 600 MHz NMR spectrometer equipped with a 5 mm CPTCI cryoprobe was used to acquire NMR data at 310 K (37 ◦C) Bruker Biospin Corp, Billerica, MA, USA). Samples were prepared in 5 mm step-down NMR tubes (Wilmad LabGlass, Vineland, NJ, USA) with 5% D2O. A modified 2D gradient-selected, sensitivity-enhanced 1H-13C HSQC NMR method [14] with additional excipient signal suppression was used to acquire the methyl fingerprints of the samples. The WET scheme [15] was used to suppress the acetate signal, and the asymmetric adiabatic pulse (HS1/2, R = 10, 0.9 Tp;

tanh/tan, R = 50, 0.1 Tp), with pulse length 375 μs [16], was applied to suppress the carbon signals of the sucrose while exciting the methyl 13C signals of the protein. 2D 1H-13C HSQC experiments for Figure 3 used the following parameters to acquire NMR data: The f2 spectral width was 14 ppm centered on 4.7 ppm with 2048 points. The f1 spectral width was 28 ppm centered on 21 ppm. Spectra were acquired with 128 increments with 50% non-uniform sampling and 2048 scans in each increment, with recycle delay 0.5 s between scans. The total experimental time was 26.5 h for each spectrum. Digital filtering for 0.4 ppm bandwidth was used to further remove the water signal. GARP decoupling was applied during the WET scheme with 2.08 kHz RF power and the t2 acquisition with 4.16 kHz RF power. Shifted sine-squared bell window functions and zero filling were applied to both dimensions before Fourier transform of the data. The final spectra were 4 k × 1 k. The spectrum in Figure <sup>4</sup> was acquired using the 1H-13C multiplicity-edited HSQC (hsqcedetgpsisp2.2 in the Bruker library). The f2 spectral width was 9 ppm, centered on 4.5 ppm with 2048 points. The f1 spectral width was 160 ppm centered on 80 ppm. The 2D data were obtained with 512 increments and 16 scans in each increment, with recycle delay 1 s between scans. The total experimental time was 2.8 h. The data processing was carried out using the spectrometer software (TopSpin, Bruker BioSpin Corp, Billerica, MA, USA) and Mnova software (Mestrelab Research S.L., Santiago de Compostela, Spain).
