Avance 700

All ¹H-NMR spectra were acquired using a Bruker Avance 700 MHz spectrometer equipped with a triple resonance TXI probe and a SampleXpress Lite autosampler. All spectra were acquired at 25 °C using noesypr1d sequence, 1024 scans, 4 dummy scans, a spectral width of 16 ppm, an acquisition time of 3 s, a relaxation delay of 3 s, and a mixing time of 100 ms.
All the spectra were processed using 0.5 Hz of line-broadening followed by manual phase and baseline correction. Chenomx NMRSuite 8.5 (Chenomx Inc., Edmonton, AB, Canada) was used to quantify the metabolites. The spectra database in this software allows a manual deconvolution of the different signals and determines the concentration of the compounds that form the mixture. TSP was set as an internal standard, and 53 metabolites were quantified in almost all samples (missing values 5%). Concentration values of pyroglutamate were summed to those of glutamine as the cyclization reaction of glutamine to pyroglutamate occurs during protein removal [19 (link)].

¹H NMR measurements were performed on a Bruker AVANCE-700 (Bruker, Rheinstetten, Germany) spectrometer operating at 700.13 MHz for ¹H. All spectra were recorded at 310 K using a 5-mm inverse probe and the sample volume was 450 μl in all cases. All spectra were corrected for baseline and phase distortions and calibrated using the residual proton resonance of methanol at 3.49 ppm. The lipid composition of the samples was determined by integrating the methyl (0.9 ppm), allylic (2.7 ppm), olefinic (5.3 ppm), and vicinal-olefinic (2.0 ppm) resonances.

Chemicals were purchased from commercial sources (like Sigma‐Aldrich, TCI, AlfaAesar, ABCR and Fluka). Indolenine precursors and the protected DOTA chelator were synthesized according to published procedures. ¹H, ¹³C and ⁷¹Ga NMR spectra in solution were measured with the spectrometers ECX 400 (400 MHz) and ECP 500 (500 MHz) from JEOL and Avance 500 (500 MHz) and Avance 700 (700 MHz) from Bruker. Mass spectra were measured on a 6210 ESI‐TOF and 6230 ESI‐TOF from Agilent. UV/VIS spectra were recorded using a PerkinElmer LAMBDA 950 UV/Vis/NIR spectrometer and fluorescence spectra recorded using a JASCO FP‐6500 spectrometer. NP automated column chromatography was done on a CombiFlash Rf (Teledyne ISCO) using prepacked silica columns (30 μm). Purification of compounds using size exclusion chromatography was done on a Sephadex column (NAP‐25, Sephadex G‐25 DNA) with water as eluent.

NMR spectroscopy experiments were performed using the Bruker Avance 700, 800 and 900 MHz spectrometers at the Korea Basic Science Institute (Ochang, Korea). 0.4–0.5 mM of the ACP samples was prepared in 330 μL of 9:1 (v/v) H₂O/D₂O 25 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 6.1) containing 5 mM CaCl₂ and 5 mM dithiothreitol (DTT). 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) was used as an internal chemical shift reference. 0.02% of NaN₃ was added as an antiseptic. Triple resonance spectra of HNCO, HNCACB, and CBCA(CO)NH experiments were acquired to assign the resonances of spins within the backbones of Tm-ACP. For side chain assignment, CC(CO)NH, HBHA(CO)NH, H(CCO)NH, and HCCH-TOCSY spectra were obtained. The assignments were confirmed by ¹H-¹⁵N-¹H and ¹H-¹³C-¹H NOESY-HSQC spectra [18 (link),59 (link),60 (link),61 (link),62 (link)]. All NMR spectra were processed with NMRPipe [63 (link)] and analyzed with NMRFAM-Sparky [64 (link)]. Residual dipolar coupling (RDC) constants between two spins in backbone amide N-H bonds were determined by comparing spatially anisotropic dipolar couplings in IPAP-HSQC spectra of solution and gel phase Tm-ACP sample. The gel phase sample was prepared by dissolving the solution sample in a radially compressed polyacrylamide gel [18 (link),62 (link),65 (link),66 (link),67 (link)].

A 0.5 mM sample of ¹³C,¹⁵N-labeled Orf63 was prepared for NMR spectroscopy in NMR buffer supplemented with 10% D₂O. All experiments were performed at 310 K using a Bruker Avance 700 MHz NMR spectrometer equipped with a cryogenically cooled 5 mm probe at the York University Life Sciences Building Central Facility. Backbone (HN, N, CA, CB, C') assignments were achieved using a set of conventional triple resonance experiments (HNCA, HNCACB, CBCAcoNH, HNCO, HNcaCO) incorporating sparse sampling for the optimum sensitivity and resolution. Datasets were processed with NMRpipe (Delaglio et al., 1995 (link)) and istHMS (Hyberts et al., 2012 (link)) and interpreted with CCPN Analysis (Skinner et al., 2015 (link)).