850 mhz nmr spectrometer

For assignment of paxillin-γ LIM4 apo-form,
¹⁵N/¹³C-labeled paxillin-γ LIM4
(541–605) was purified and the N-terminal GST-tag was removed during
purification. Standard triple-resonance experiments were conducted with 1.45
mM ¹⁵N/¹³C-labeled paxillin-γ LIM4 on a Bruker
850 MHz NMR spectrometer at 25 °C in the buffer containing 50 mM
NaH₂PO₄/Na₂HPO₄ (pH 6.8), 50
mM NaCl, 0.5 mM TCEP and 5% D₂O. LIM4 sequential assignment was
performed using PASA software(Xu et al.,
2006). For assignments of the bound paxillin-γ LIM4 and
kindlin-2 F0 forms, the chemical shifts of the majority of residues were
readily transferred from the free forms with sight adjustments. The
assignment of free form kindlin-2 F0 published previously by our lab (Perera et al., 2011 (link)) was downloaded
from Biological Magnetic Resonance Data Bank (accession code 17827). All
assignments were also verified by 3D-NOESY experiments of the complex listed
below.

Optical rotations were measured on a JASCO P-2000 polarimeter and UV spectra with a JASCO V-650 spectrophotometer. ECD spectra were measured on a JASCO J-815 spectrometer. The ¹H and ¹³C NMR spectra were recorded on INOVA-500 and BRUKER AV500-III spectrometers. HETLOC and HSQC with decoupling were measured on a Bruker 850 MHz NMR spectrometer. The standard Bruker pulse sequences were used for each 2D measurement. HRESIMS data were acquired using an Agilent 6520 Accurate-Mass Q-Tof LC/MS mass spectrometer. Analytical reversed-phase HPLC was performed on a COSMOSIL 5C₁₈-PAQ Waters column (4.6 mm I. D. × 250 mm) eluted with water/methanol (flow rate, 1 mL/min; 220 nm UV detection) at room temperature. Preparative reversed-phase HPLC was performed on a COSMOSIL 5C₁₈-PAQ Waters column (10 mm I. D. × 250 mm) at room temperature. Column chromatography was performed with silica gel (40–63 µm; Silicycle), C₁₈ 120 Å reversed-phase silica gel (RP-18; 50 µm; YMC), and Sephadex LH-20 (GE Healthcare Bio-Science AB). Fractions were monitored by TLC and spots were visualized by heating silica gel plates sprayed with 10% H₂SO₄ in ethanol.