Avance 500 mhz spectrometer

The NMR samples used for DNA titration experiment all contained 0.1 mM uniformly ¹⁵N labeled protein in 50 mM sodium phosphate, 50 mM NaCl (pH 6.0) with 90% H₂O/10% D₂O. A series of 2D ¹H-¹⁵N HSQC spectra with gradually increased DNA concentration (0.02 mM, 0.04 mM, 0.08 mM, 0.12 mM, 0.16 mM and 0.2 mM) were collected at 298 K on a Bruker Avance 500 MHz spectrometer with a triple-resonance cryoprobe and the chemical shifts changes were analyzed.
2D ¹H NOESY experiments were performed to investigate the chemical shift perturbations for the DNA duplex d(CGCATATATGCG)² samples with or without MvaT_ctd on a Bruker Avance 800 MHz spectrometer equipped with cryoprobe at 298 K. The NMR sample contained 0.4 mM DNA in 50 mM sodium phosphate, 50 mM NaCl (pH 6.0) with 100% D₂O, and lyophilized protein powder of MvaT_ctd was added to final concentrations of 0.25 mM and 0.5 mM. The fingerprint region of intraresidual H1’-H6/H8 NOE cross peaks was analyzed.
For netropsin competition experiment, 0.1 mM uniformly ¹⁵N labeled MvaT_ctd was mixed with 0.2 mM DNA duplex in 50 mM sodium phosphate, 50 mM NaCl (pH 6.0) with 90% H₂O/10% D₂O. 2D ¹H-¹⁵N HSQC spectra were collected with gradual addition of netropsin at concentrations of 0.05 mM, 0.1 mM, 0.2 mM, 0.3 mM and 0.5 mM, on a Bruker Avance 500 MHz spectrometer with a triple-resonance cryoprobe.

FTIR (Fourier transform infrared) spectra were recorded on a Nicolet iS5 Mid Infrared FT-IR Spectrometer equipped with iD7 ATR optical base (Thermo Scientific®, Waltham, MA, USA). ¹H, ¹³C, COSY (correlation spectroscopy) and HSQC-DEPT (heteronuclear single quantum correlation-distortionless enhancement by polarization transfer) NMR spectra were recorded on a Varian VXR 400 MHz (Palo Alto, Ca, USA) or Bruker AVANCE 500 MHz spectrometers (Bremen, Germany) using tetramethylsilane as an internal standard and deuterated solvents (CDCl₃, DMSO-d₆). Samples of 60–100 mg per 0.5 mL of the solvent were used. The average molecular masses of polymers were determined based on ¹H NMR spectra. The integral of the CH₃ group signal coming from the core trimethylolpropane (TMP) molecule was used as a reference. Elemental analysis was performed with Elementar Vario EL III CHNS analyzer (Analysensysteme GmbH, Hanau, Germany). DTA/TG measurements were carried out by using Netzsch Jupiter STA 449C coupled with a Netzsch QMS 403C Aeolos mass spectrometer (Selb, Germany). The heating rate was 10 °C min⁻¹ and the final temperature was 600 °C. The measurements were performed in the constant flow of two gases: argon—10 mL min⁻¹ (protective gas) and synthetic air (80:20 N₂:O₂)—60 mL min⁻¹.

Bruker Avance 500 MHz spectrometers (Bruker Corporation, Karlsruhe, Germany) were used to record all NMR spectra using methanol-d₄ as solvent. Chemical shifts were reported with reference to the residual solvent peak δ_H 3.31 and δ_C 49.0 ppm. Waters LCT Premier XE (Waters, Sydney, NSW, Australia) time-of-flight mass spectrometer was used for high-resolution mass spectra in positive electrospray ionization mode. Apollo reversed phase C18 column (250 mm × 10 mm, 5 μM, Grace-Davison Discovery Sciences, Melbourne, VIC, Australia) was used for separation in semi-preparative HPLC (Agilent Series 1200). HPLC was equipped with a photodiode array detector and preparative fraction collector.