Avance 850 mhz

To generate ¹³C/¹⁵N-enriched PI3Kα RBD, pCDB24 construct containing our gene of interest, was expressed in BL21(DE3)-RIPL E. coli bacterial strain in minimal medium [41 (link)] containing 1 g/L ¹⁵NH₄Cl and 3g/L ¹³C-glucose (Cambridge Isotope Laboratories) as the sole source of nitrogen and carbon, respectively. The cells were grown at 37°C to an OD₆₀₀ ~ 0.8 and induced with 500 μM IPTG followed by incubation at 18°C. After 22 h, the cells were harvested at 4 °C and 5000 x g and then purified as described above. ¹³C/¹⁵N-enriched PI3Kα protein (157 μM) was equilibrated in 20 mM HEPES (pH 6.8), 50 mM NaCl and 5 mM DTT containing 5% (v/v) D₂O. Two-dimensional NMR ¹H-¹⁵N HSQC spectrum of ¹³C/¹⁵N-labelled PI3Kα RBD was acquired on a Bruker Avance 850 MHz (19.97 T field strength) spectrometer at 25 °C, using a cryogenic (TCI) 5 mm triple-resonance probe equipped with z-axis gradient. NMR data was collected using a spectral width of 13 ppm and 33 ppm and complex points of 1024 and 256 along the ¹H and ¹⁵N dimension, respectively. The NMR data was processed using NMRPipe [42 (link)] and the spectrum was visualized using SPARKY [43 (link)].

NMR experiments were acquired with either a Bruker Avance 700 MHz spectrometer equipped with a 5 mm TCI cryoprobe or a Bruker Avance 850 MHz spectrometer equipped with a triple resonance TXI probe. All experiments were acquired at 306 K unless indicated otherwise. Multidimensional experiments were processed using the NMRpipe suite of programs and analyzed in SPARKY.^{78 (link),79 (link)} One-dimensional experiments were processed and analyzed directly in Topspin. Additional details about data acquisition, processing, and analysis are described below.

To generate ¹⁵N-enriched KRAS^Q61E, the protein was
recombinantly expressed as above, using minimal media containing 1 g/L
¹⁵NH₄Cl (Cambridge Isotope Laboratories) as the sole
source of nitrogen. Purification of ¹⁵N-enriched KRAS^Q61Erequired no modifications to the purification protocol described above. To
produce the nonhydrolyzable GTP analog GMPPCP-bound KRAS^Q61E protein,
nucleotide loading was performed utilizing the alkaline phosphatase bead
incubation method, as described above. Nucleotide loading was verified via HPLC
analysis to exceed 95% for the desired state. For NMR analysis,
¹⁵N-enriched KRAS^Q61E (100 μM) was equilibrated in
a buffer containing 20 mM NaH₂PO₄ (pH 6.8), 50 mM NaCl and
5 mM MgCl₂, supplemented with 5% (v/v) D₂O.
Two-dimensional NMR ¹H-¹⁵N HSQC spectra of
¹⁵N-labelled KRAS^Q61E were acquired on a Bruker Avance 850
MHz (19.97 T field strength) spectrometer at 25°C, using a cryogenic
(TCI) 5 mm triple-resonance probe equipped with z-axis gradient. NMR data were
collected using a spectral width of 16 ppm and 38 ppm and complex points of 2048
and 128 along the ¹H and ¹⁵N dimension, respectively. The
NMR data were processed using TopSpin (v3.6.1, Bruker) and the spectra were
visualized using SPARKY (57 (link)). These NMR
data were collected on both the inactive GDP- and active GMPPCP-bound
states.