Drx 500 spectrometer

IR spectrum was recorded on a Nicolet 6700 spectrometer (Thermo Scientific, Waltham, MA, USA). Optical rotation was measured on a PerkinElmer 241 MC polarimeter (PerkinElmer, Fremont, CA, USA). ¹H and ¹³C NMR spectra were acquired on a Bruker DRX-500 spectrometer (Bruker Biospin AG, Fällanden, Germany). Chemical shifts (δ) are reported in ppm with reference to the solvent signals, and coupling constants (J) are in Hz. ESIMS spectra were recorded on a Finngan-MAT-95 mass spectrometer and HRESIMS spectra were recorded on an Agilent 1290–6545 UHPLC-QTOF mass spectrometer. Commercial silica gel (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China, 200–300 and 300–400 mesh) and Sephadex LH-20 gel (Amersham Biosciences, Little Chalfont, UK) were used for column chromatography (CC). Precoated silica gel GF-254 plates (Sinopharm Chemical Reagent Co., Shanghai, China) were used for analytical TLC. Reversed-phase (RP) HPLC was performed on an Agilent 1260 series liquid chromatograph equipped with a DAD G1315D detector at 210 nm (Agilent, Santa Clara, CA, USA). An Agilent semipreparative XDB-C18 column (5 μm, 250 × 9.4 mm) was employed for the purification. All solvents used for CC and HPLC were of analytical grade (Shanghai Chemical Reagents Co., Ltd., Shanghai, China) and chromatographic grade (Dikma Technologies Inc., Lake Forest, CA, USA), respectively.

All samples for NMR spectroscopy were prepared at concentrations of 200–800 μM in phosphate buffer supplemented with 10% D₂O (Sigma) and 0.0025% 3,3,3-trimethylsilylpropionate (Sigma) in 5 mm Ultra-Imperial grade NMR tubes (Wilmad) to a final volume of 600 μL. 10 mM DTT was added to holo samples. [¹H,¹⁵N]-HSQC, ¹⁵N-TOCSY-HSQC, ¹⁵N-NOESY-HSQC, HNCA, HNCOCA, HNCACB and CBCA(CO)NH spectra were recorded at 283 K on a Bruker DRX500 spectrometer equipped with a z-shielded gradient triple resonance probe, using standard procedures [29 ]. ¹³C-NOESY-HSQC spectra were recorded at 283 K on a Bruker Avance DRX800 spectrometer equipped with a 5 mm TXI CryoProbe. All NMR spectra were processed using the Azara package (www.ccpn.ac.uk/azara), then analysed and assigned using CcpNmr Analysis software [30 (link)]. To compare resonance positions in [¹H,¹⁵N]-HSQC spectra of different mACP₉ species, average chemical shift differences were determined using the formula Δδ_av={0.5(Δδ_H)² + 0.1(Δδ_N)²}^0.5 [31 (link)]. The threshold for a significant shift change (0.042 ppm) was calculated as twice the S.D. of the differences in all data sets remaining after eliminating outliers with differences greater than two S.D. from the initial mean.

¹⁵N nuclear spin relaxation experiments were recorded using standard procedures [29 ] at 283K on a Bruker DRX500 spectrometer. ¹⁵N T₁ delays (ms): 10, 50, 100, 150, 250, 400, 550, 700, 850, 1000. ¹⁵N T₂ delays (ms): 14.4, 28.8, 43.2, 57.6, 72.0, 86.4, 100.8, 155.2. The heteronuclear NOE reference and saturation experiments were carried out in duplicate to allow an estimation of the error. An initial τ_c estimate was obtained from the R₂/R₁ ratios for each residue [37 (link)]; the same procedure was used to make site specific estimates of the local rotational correlation time τ_eff. The relaxation parameters were analysed with version 4 of the Modelfree program [38 (link)] using the strategy described by Mandel et al. [39 (link)]. H^N─N bond vectors from the solution structure of apo mACP₉ were used for anisotropic diffusion tensor modelling of relaxation data for both the apo and holo forms.

GC measurements were performed on an HP5890 Series 2 GC-FID chromatograph, using a 15 m × 0.18 mm Restek, Rtx-5 column with a film layer of 0.20 μm. The temperature of the column was initially held at 40 °C for 1 min, followed by programming at 25 °C/min up to 300 °C and a final period at 300 °C (isothermal) for 10 min. The temperature of the injector was 290 °C and of the FID detector was 300 °C. The carrier gas was N₂.
GC-MS measurements were performed on an Agilent 6890 N-GC-5973 N-MSD chromatograph, using a 30 m × 0.25 mm Restek, Rtx-5SILMS column with a film layer of 0.25 μm. The initial temperature of the column was 45 °C for 1 min, followed by programming at 10 °C/min. up to 310 °C and a final period at 310 °C (isothermal) for 17 min. The temperature of the injector was 250 °C. The carrier gas was He, and the operation mode was splitless.
High resolution mass spectrometric measurements were performed using a TripleTOF 5600+ mass spectrometer in positive electrospray mode.
The ¹³C- and ¹H-NMR spectra were obtained in CDCl₃ solution on a Bruker DRX-500 spectrometer operating at 125.7 and 500.1 MHz, respectively. The ¹³C and ¹H chemical shifts are referred to TMS. ³¹P-NMR spectra were obtained on a Bruker AV-300 spectrometer at 121.5 MHz. Chemical shifts are downfield relative to 85% H₃PO₄.