TROSY NMR with a 52 kDa sugar transport protein and the binding of a small-molecule inhibitor

Figures & data

Figure 1. Stability of purified GalP reconstituted in DDM micelles. (A) Overlay of far-UV circular dichroism spectra for purified GalP [10 μM in 10 mM KPi buffer, pH 7.5, 5% glycerol, 0.05% DDM] recorded at a temperature of 25 °C using slit widths of 0.5 mm with repeated scans every 20 min over a period of 48 h (left) and plots of the CD signal at wavelengths of 193, 210 and 223 nm against time (right). The black dashed spectrum is from a separate sample that was subject to rapid degradation over 100 min by exposure to significantly higher intensity UV light using slit widths of 1 mm. (B) Fluorescence spectra excited at 280 nm for purified GalP [100 μg/ml in KPi buffer (10 mM, pH 7.5), 5% glycerol, 1% DDM] recorded at a temperature of 25 °C following successive additions of the inhibitor cytochalasin B (to give concentrations of 0, 1, 2, 3, 5, 10, 15, 20, 30, 40, 50 μM) on aliquots removed at a number of time-points from a solution of protein held at a temperature of 25 °C (top). This experiment was repeated on protein samples held at a range of temperatures and the % quench in fluorescence intensity at 330 nm on titration with cytochalasin B used as a measure of protein activity (bottom).

Figure 2. Amplified expression and purification of tryptophan-labelled GalP and detection of signals for all 12 tryptophan residues. (A) Coomassie-stained SDS-PAGE analysis for amplified expression of [U-²H, ¹⁵N₂-Trp]GalP(His₆) in native membranes (left) and the purified protein reconstituted in [²H]DDM micelles (right). (B) [¹⁵N-¹H]TROSY spectrum at 900 MHz of [U-²H, ¹⁵N₂-Trp]GalP(His₆) (∼165 μM) in [U-²H]DDM (1%), [²H₈]glycerol (5%), KPi buffer (10 mM, pH 7.5) acquired over 65 h at 25 °C.

Figure 3. Improvement in resolution for detection of isoleucine, leucine and valine methyl groups at 900 MHz over 750 MHz. [¹³C-¹H]-Methyl-TROSY spectra at 750 MHz (left) and 900 MHz (right) of [U-²H, U-¹⁵N, Ile-D₂, ¹³CH₃, Leu/Val-¹³CH₃, ¹²CD₃]GalP(His₆) (51.8 μM) in DDM (0.05%), d₈-glycerol (5%), D₂O (10%), KPi buffer (10 mM, pH 7.5) acquired over 21 h at 20 °C. The asterisk (*) indicates a signal coming from the detergent DDM.

Figure 4. Detection of inhibitor binding to GalP in methyl-TROSY spectra at 900 MHz. (A) Ile C^δ1 region of [¹³C-¹H]-methyl-TROSY spectra at 900 MHz of [ILV-¹³CH₃]GalP(His₆) (51.8 μM) in DDM (0.05%), d₈-glycerol (5%), D₂O (10%), KPi buffer (10 mM, pH 7.5) in the absence (black) and presence (red) of forskolin (100 μM with 1% d₆-ethanol) acquired over 21 h at 20 °C. The largest chemical shift changes are indicated with numbered arrows. The asterisk (*) indicates a signal coming from the detergent DDM. (B) Histogram of chemical shift change metric (SQRT[(3*Δ¹H)²+ (Δ¹³C)²]) ordered according to the size of the chemical shift change upon binding forskolin for the Ile C^δ1 peaks. Changes for signals beyond those labelled 1–5 are considered as not significant.