NMR
Triple-resonance and NOESY spectra were collected on 0.1-1 mM protein
samples in PBS using either a 600 or 800 MHz Varian DD2 console equipped
with cryogenically cooled probe. Backbone and side-chain assignments,
and NOESY distance restraints were obtained using standard triple
resonance techniques employing either the standard VARIAN Biopack or
modified BEST-TROSY pulse sequences [24, 25]. T1 and
T2 relaxation times were obtained for the1H-15N peaks using a modified HSQC
pulse sequence, and used to determine Tc as described
previously [46].
Epitope prediction and ΔSIM values were obtained using the SPADE
(Surface comparison-based Prediction of Allergen Discontinuous Epitopes)
algorithm access via the provided web server. NMR structures A1Pro and
the J2LS fragments were used as inputs. Predicted cross-reactivity was
listed as 55% between A1LS and the J2LS fragments, or 100% within the
JR22 fragment. These values were selected to broadly reflect the
cross-reactive IgE binding observed in the microarray studies[29,
47].