NMR Characterization of PIRT and CaM

¹⁵N-human PIRT (180 μL, 3 mm NMR tube) and ¹⁵N-human CaM (550 μL, 5 mm NMR tube) were measured in NMR buffer (4% D₂O (v/v, Sigma Aldrich), 20 mM sodium phosphate (Fisher Scientific), 500 µM DSS (Sodium trimethylsilylpropanesulfonate, Sigma Aldrich), and 0.5 mM EDTA (Sigma Aldrich) at pH 6.5) on a Bruker 850 MHz ¹H magnet with Avance III console. Two thousand forty-eight direct points and 128 indirect points were collected with 128 transients, processed in NMRpipe [47 (link)], and analyzed in CCPNMR [48 (link)]. Optimization of NMR conditions for PIRT was carried out previously [24 (link)], resulting in dodecylphosphocholine (DPC) as the most suitable detergent for investigations with PIRT and at a temperature of 40 °C. An HSQC of ¹⁵N-human CaM was measured with and without CaCl₂ to show that it was properly folded in the conditions we tested.
To validate the cholesterol-like and β-estradiol binding site, a saturating concentration of β-estradiol (3.82 mole %) was dissolved in DMSO before adding to a ¹⁵N-human PIRT sample. An HSQC of human PIRT was measured before and after adding β-estradiol, and the resonances with significant chemical shift perturbation identify the amino acids that comprise the binding site.