Avance 3 hd 800

NMR measurements for rSslE were performed at 25 °C on a 100 µM ²H¹⁵N-labelled sample in 50 mM NaPO₄ pH 7.4, 100 mM NaCl, 10 % D₂O. NMR measurements for SslE NT1 (0.6 mM), SslE NT2 (1.3 mM) and SslE NT1-NT2 (0.8 mM) were performed at 25 °C on ¹⁵N-labelled samples in 50 mM NaPO₄ pH 7.0, 100 mM NaCl, 10 % D₂O. NMR measurements for RgpB-CTD (0.3 mM) were performed at 37 °C on a ¹⁵N-labelled sample in 20 mM NaPO₄ pH 6.0, 100 mM NaCl, 10% D₂O. Transverse relaxation optimised spectroscopy (TROSY) based ¹H¹⁵N-HSQC experiment and T₁ and T₂ relaxation times for rSslE were recorded on a Bruker Avance III HD 950, equipped with a TXI cryoprobe. TROSY ¹H¹⁵N-HSQC experiment for SslE NT1-NT2 was recorded on a Bruker Avance III HD 800, equipped with a TCI cryoprobe. Standard ¹H¹⁵N-HSQC spectra of SslE NT1, SslE NT2 and RgpB-CTD were recorded on a Bruker Avance III HD 700, equipped with a TCI cryoprobe. Data were processed in NMRPIPE^{65 (link)} and analysed/visualised with ANALYSIS^{66 (link)} and NMRVIEW^{67 (link)}.

NMR experiments for resonance assignment were performed on a Bruker Avance III HD 800-MHz spectrometer equipped with a cryogenically cooled probe head. All samples were prepared in a buffer containing 20 mM Sodium Phosphate pH 6.3, 100 mM KCl, and 5% (v/v) D₂O. The NMR spectra for backbone resonances assignments were collected using a 450 μM uniformly ¹³C, ¹⁵N–labeled DrFoxM1 TAD protein. NMR labeled TAD protein was phosphorylated by Plk1 kinase domain as described above. Sequence-specific backbone resonance assignments for DrFoxM1 TAD were determined using HSQC, HNCO, HNCACB, CBCA(CO)NH, and C(CO)NH experiments supplied by Bruker BioSpin. Sequence-specific backbone resonance assignments for phosphorylated DrFoxM1 TAD were determined using HSQC, HNCO, HNCACB, and CBCA(CO)NH experiments supplied by Bruker BioSpin.

¹H-NMR spectra of the CVF samples were acquired on a Bruker Avance III HD 800 MHz FT-NMR spectrometer using a 5 mm triple-resonance inverse (TCI) cryoprobe with Z-Gradients (Bruker BioSpin Co., Billerica, MA, USA). To acquire one-dimensional (1D) 1H spectra of the CVF samples, Bruker standard 1D 1H T2 filter (Car–Purcell–Meiboom–Gill (CPMG)) pulse sequence was used with the relaxation delay (RD) = 2.0 s, CPMG echo delay (τ) = 0.2 ms, repetitions number (n) = 256, dummy scans = 16, loops = 160, and acquisition time (Acq) = 2.0 s. The water signal was suppressed at the water peak during RD. Free induction decay (FID) was acquired with a spectral width of 20 ppm for 64,000 data points.
As the reference sample, an ERETIC (electronic reference to access in vivo concentrations) reference (ER) sample was used [53 (link)]. In the ER sample, valine was included as a reference molecule. The reason why valine was used as a reference molecule is that the methyl group of valine does not interact with serum macromolecules such as human serum albumin (HSA) and fatted HSA (fHSA). Thus, the ER sample was prepared by mixing 150 μL of CVF, 12 μL of 100 mM valine, and 438 μL of D2O, to achieve a final valine concentration of 2 mM.