Figure S1Figure S1. Correlation of mite ImmunoCAP scores with S6-sIgE. For the 36 patients for whom data were available, the mite ImmunoCAP score was compared with the measured S6-sIgE, which is the sum of the specific IgE to Der p 1, Der p 2, Der p 5, Der p 7, Der p 8, and Der p 23. The correlation coefficient is 0.67. Lines indicate the geometric mean of the S6-sIgE for each score group.

Blo t 12 81 --------------------DLIVHEGGKTYHVVCHEEGPIHIQEMCNKYIICSKSGSLWTachychitin 1 --------------------YL-AFRCG--RYSPCLDDGPNVNLYSCCSFYNCHK----CrDer p 23 21 -------------------SFT-KFECP--SRFGYFADP-----KDPHKFYICSN----WDer p 23-Nterm 2 ANDNDDDPTTTVHPTTTEQPDD .. : .: * :

Blo t 12 121 YITVMPCSIGTKFDPISRNCVLDN-----------------Tachychitin 34 LARLENCPKGLHYNAYLKVCDWPSKAGCTSVNKECHLWKTXrDer p 23 50 EAVHKDCPGNTRWNEDEETCT-------------------- * ::: . *

Figure S2

Figure S2. Alignment of Blo t 12, Tachychitin, rDer p 23, and the N- terminus of full length Der p 23. Dots and asterisks below indicate the level of conservation according to BLAST. The red coloring indicates beta strands. The green coloring shows residues introduced to facilitate TEV cleavage, and blue is the N-terminus of full length Der p 23.

CBP

rDer p 23

MBP

I S E

Figure S3 Figure S3. Binding experiments with chitin beads. Coomassie stained PAGE showing Chitin binding protein (CBP), rDer p 23, and maltose binding protein (MBP). The initial sample is in column I, the supernatant after incubation with the beads is column S, and the eluent is in column E. CBP demonstrated a positive control and that the column capacity was not overloaded. rDer p 23 did not stick to the chitin beads and remained in the supernatant, similar to MBP, which served a negative control.  Method Notes: Chitin beads were purchased from New England Biolabs. A 400 ml slurry of 50% beads was washed 5 times with 200 ml volumes of PBS. Samples were added at protein concentrations of 1-2 mg/ml, which is equal to the column protein capacity. Chitin binding protein was added unpurified from bacterial extract. Der p 23 and MBP were previously purified (as above). The samples were incubated with the beads for 1-2 hours with gentle agitation, followed by 5 washes with 200 ml of PBS. Protein elution from the beads was accomplished by adding 200 ml of SDS gel loading sample buffer with 100 mM DTT and incubating at least 5 minutes at 95 C. Proteins were detected on SDS PAGE with Coomassie stain.

A51

Figure S4

Figure S4. NMR experiments with rDer p 23 and a chitin fragment. Four HSQC spectra of 15N labeled rDer p 23 are compared. The protein was 100 mM and the chitin fragment beta-1,4 linked di-N-acetyl glucosamine (Omicron Biochemicals) was 0 (black), 1 mM (red), 2 mM (blue), 3mM (green). The bottom panel zooms in on the amide of A51 to demonstrate the coincidence of all 4 spectra. The lack of changes in amide chemical shifts indicates a lack of binding to the substrate.

Glucose

Cellobiose (b-1-4)

N-acetyl-D-glucosamine

STD-1

STD-2

STD-2

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Figure S5

Figure S5. Saturation Transfer Difference NMR experiments [1]. 1H NMR spectra of Glucose (red), Cellobiose (orange), and N acetyl-D-glucosamine (blue). Chemicals were purchased from Sigma. STD NMR experiments of mixed glucose and cellobiose (magenta and green) at two different saturation frequencies indicated by arrows. STD NMR experiments of N acetyl-D-glucosamine (cyan and purple) at two different saturation frequencies indicated by arrows. Der p 23 was 50 mM and substrates were at 1 mM. Line drawings of the small molecules are included. In this experiment, the substrate spectra (red, orange, or blue) should be reproduced in the STD experiment (magenta, green, cyan, or purple) if binding occurs. Two saturation frequencies were tested for a mixture of glucose and cellobiose, and N-acetyl glucosamine alone. No experiment appeared to show any ligand signal in the STD indicative of binding to these compounds. Because the rDer p 23 is so small, there is some protein signal that is not completely canceled that depends on the saturation frequency (indicated by the arrows), but this signal is not indicative of any binding of ligands to rDer p 23. 1. Mayer M, Meyer B. Characterization of ligand binding by saturation transfer difference NMR spectroscopy. Angew Chem Int Edit. 1999; 38(12):1784-8.

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Phi Correlations

Xtal

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Psi Correlations

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Figure S6

A) r=0.88 B) r=0.99

Figure S6. Comparison of crystal versus solution phi/psi angles. Panels A) and B) show the correlation of the Phi and Psi angles respectively measured in the crystal structure versus the prediction of the same angle from NMR chemical shift data.