Ligand Recognition of the Major Birch Pollen Allergen Bet v 1 is Isoform Dependent
Fig 2
Binding of naringenin to Bet v 1a.
A, UV/VIS spectra of the equilibrium titration of 20 μM naringenin with Bet v 1a. All spectra were recorded at 298 K with 50 mM sodium phosphate, 50 mM NaCl at pH 7.0 and 10% DMSO as sample buffer. B, Absorbance changes at 325 nm plotted against the Bet v 1a concentration as shown for the data in A. The curve represents the best fit to Eq (1) resulting in a Kd value of 60.6 ± 3.2 μM. C, Overlay of six 1H-15N HSQC spectra of 100 μM Bet v 1a in the presence of increasing naringenin concentrations from light to dark red. The experiments were performed with a Bruker Avance 700 MHz spectrometer in 50 M sodium phosphate, 50 mM NaCl, pH 7.0 and 10% 2H2O at 298 K. Naringenin was added from a stock prepared in deuterated DMSO to a final excess of 1:4.5 over Bet v 1a and a final DMSO concentration of 10%. D, Chemical shift changes (Δδnorm) calculated with Eq (2) for residues A15 (○) and G89 (●) plotted against the ration of naringenin:Bet v 1a during titration. The curves represent the best fit to a quadric binding equation from the analysis software of NMRViewJ [89] (S1 Table). E, Calculated Δδnorm values upon naringenin addition plotted against the Bet v 1a amino acid sequence and F, mapped on a cartoon representation of the complex structure of Bet v 1a:naringenin (pdb code: 4A87) with 0.04 ppm ≤ Δδ ≤ 0.08 ppm shown as yellow; 0.08 ppm ≤ Δδ ≤ 0.12 ppm shown as orange; and 0.12 ppm < Δδ shown as red. Bet v 1a in grey, naringenin in green sticks, oxygen in red.