1h 13c 15n tci cryogenic probe head

NMR measurements were performed in a 600 MHz Bruker Avance III spectrometer equipped with a 1H/13C/15N TCI cryogenic probehead with z-axis gradients at the CEITEC Josef Dadok National NMR Centre. Chemical shift assignments were obtained using the 4D-CHAINS technology^{84 (link)}. For titration experiments, ${}^1\text{H}$ – ${}^{15}\text{N}$ HSQC spectra of $100\mathrm{\mu }\text{M}$ PDZ wt/mutant solution were recorded with increasing amounts of C-terminal DVL peptide consisting of aa 698-716 from DVL3 with phosphorylated pS700 (stock concentration of $800\mathrm{\mu }\text{M}$ ). The peptides were uncapped. The weighted ${}^1\text{H}$ and ${}^{15}\text{N}$ chemical shift differences were calculated according to the following equation $$\begin{array}{c}\hfill \mathrm{\Delta }{\delta }^{\mathit{obs}}=\sqrt{{\left({\delta }^{1H}\right)}^2+{\left(\frac{1}{6},{\delta }^{15N}\right)}^2},\end{array}$$ where $\mathrm{\Delta }{\delta }^{\mathit{obs}}$ represents the observed chemical shift difference, ${\delta }^{1H}$ is the change in hydrogen chemical shift, and ${\delta }^{15N}$ is the change in nitrogen chemical shift.

A 4D HC(CC-TOCSY(CO))NH and a 4D ¹³C, ¹⁵N edited HMQC-NOESY-HSQC experiments were recorded at CEITEC Josef Dadok National NMR Centre on a 700 MHz Bruker Avance III spectrometers equipped with ¹H/¹³C/¹⁵N TCI cryogenic probe head with z-axis gradients. 4D spectra were processed with SSA package^{59 (link)} and analyzed with SPARKY. Chemical shift assignments were obtained automatically using 4D-CHAINS^{60 (link)} and checked manually. NMR titrations were performed in series of ¹H, ¹⁵N HSQC spectra using 100 μM of ¹⁵N-labeled protein (wild type or phosphorylation-mimicking variant) and increasing amounts of C-terminal DVL peptide (pS700) consisted of aa 698–716 from DVL3 (stock concentration of 800 μM). Steady-state¹⁵N-¹H Nuclear Overhauser Effect values were measured under a steady-state condition with a 30 s interscan relaxation delay. Reference spectra and the spectra measured under steady-state conditions were measured in an interleaved manner.

NMR experiments were carried out at CEITEC Josef Dadok National NMR Centre on a 700 MHz Bruker Avance III spectrometers equipped with ¹H/¹³C/¹⁵N TCI cryogenic probe head with z-axis gradients. Chemical shift assignments of the phosphomimicking PDZ mutant (S281E + S298E) were obtained automatically using 4D-CHAINS technology [37 (link)] as described before for the wt PDZ domain [24 (link)]. NMR titrations were performed in series of ¹H-¹⁵N HSQC spectra using 100 μM of ¹⁵N-labeled protein (wild type or phosphomimicking mutant) and increasing amounts of DVL_C (stock concentration of 800 μM).