Q2DSTD NMR deciphers epitope-mapping variability for peptide recognition in integrin v 6
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1 Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry. This journal is The Royal Society of Chemistry 2015 Q2DSTD NMR deciphers epitope-mapping variability for peptide recognition in integrin v 6 Jessica L. Sorge, Jane L. Wagstaff, Michelle L. Rowe, Richard A. Williamson* and Mark J. Howard* SUPPORTING INFORMATION 1 H, 13 C-HSQC WITH INVERSION RECOVERY 1 H y x,-x -x -x rec 13 C 1 t 1 dec G z All phases are x unless otherwise stated 1 = x,-x (incremented for second dimension) GARP 13 C decoupling was used during acquisition using Garp. Receiver = x,-x,x,-x (control) or x,-x,-x,x (difference) A comb of n saturation pulses are applied over the required saturation period at the desired frequency offset to provide saturation transfer (on) or not (off). Sat pulse = off,off,off,off (control) = on,on,off,off (difference) [ ] unit at the head of the sequence provides the adjustable element to measure T 1 of 1 H attached to 13 C. The inter-sequence relaxation delay was set to 5 s and values were 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 1.0 s to provide intensities for each 1 H resonance correlated to its 13 C in the modified HSQC and example intensity data are shown below for four 1 H- 13 C correlations in FMDV2:
2 Curves were fitted to: I z = I o [1 2e(-t/T 1 )] where I z is the measured intensity and t is the tau delay in 180-t-90 delay. Intensities were measured for each 2D dataset obtained with a specific tau value using CCPN Analysis. PEPTIDE 1 H, 13 C HSQC ASSIGNMENTS FMDV2
3 DBD1 LAP2T1
4 2DSTD, 1 H T 1 and Q2DSTD DATA. Intensities are in arbitrary units and only for assignable peaks with measurable 2DSTD FMDV2 2DSTD (arb units) 2DSTD % 1 H T 1 (s) Q2DSTD (arb units) Q2DSTD % 1 AsnHba AsnHbb AlaHa AlaHb ValHa ValHb ValHga ValHgb ProHa ProHb ProHb ProHd ProHd ProHg AsnHb AsnHb LeuHa LeuHb LeuHg LeuHda LeuHdb ArgHa ArgHb ArgHb ArgHd ArgHg GlyHa AspHa AspHb AspHb LeuHa LeuHb LeuHg LeuHda LeuHdb GlnHa GlnHb GlnHb GlnHg ValHa ValHb ValHga
5 12ValHgb LeuHa LeuHb LeuHg LeuHda LeuHdb AlaHa AlaHb GlnHa GlnHb GlnHb GlnHg LysHa LysHd LysHe LysHg ValHa ValHb ValHgb ValHga AlaHa AlaHb ArgHa ArgHb ArgHb ArgHd ArgHg ThrHaHa ThrHb ThrHg
6 DBD1 2DSTD (arb units) 2DSTD % 1 H T 1 (s) Q2DSTD (arb units) Q2DSTD % 2LysHe LysHg ProHa ProHda ProHdb AsnHba AsnHbb LeuHb LeuHda LeuHdb LeuHg ArgHba ArgHbb ArgHd ArgHg GlyHa AspHba AspHbb LeuHa LeuHb LeuHda LeuHdb LeuHg GlnHa GlnHba GlnHbb GlnHg ValHa ValHb ValHga LeuHa LeuHb LeuHda LeuHdb LeuHg AlaHa AlaHb GlnHa GlnHba GlnHbb GlnHg ValHa ValHb ValHga CysHba
7 18CysHbb ArgHba ArgHbb ArgHd ArgHg ThrHa ThrHb ThrHg
8 LAP2T1 2DSTD (arb units) 2DSTD % 1 H T 1 (s) Q2DSTD (arb units) Q2DSTD % 3ThrHa ThrHb ThrHgb ThrHa ThrHb ThrHgb GlyHa ArgHa ArgHb ArgHb ArgHa ArgHb ArgHb GlyHa AspHb AspHb (10Leu/16Leu)Hda (10Leu/16Leu)Hg (10Leu/16Leu)Hb (10Leu/16Leu)Hdb LeuHa AlaHa AlaHb ThrHa ThrHb ThrHgb IleHa IleHb IleHda IleHga IleHgb HisHb HisHb GlyHa LeuHa (10Leu/16Leu)Hda (10Leu/16Leu)Hg (10Leu/16Leu)Hb (10Leu/16Leu)Hdb AsnHb AsnHb ArgHb ArgHb ArgHd ProHa
9 19ProHb ProHb ProHd ProHd ProHg PheHb Q2DSTD DATA SHOWN ACROSS EACH PEPTIDE SEQUENCE FOR ALL NUCLEI Successive residues in sequence shown by alternating white then shaded backgrounds. Note the general topology of FMDV2 and DBD2 are similar and this is to be expected because they are identical sequences (except for the disulphide cyclisation in DBD2). However, the cyclisation appears to elevate N- terminal and C-terminal contacts in DBD1 as well as boost RGD-based contacts. LAP projects significant contacts across the much of the peptide sequence and it is likely these contacts are also used when binding other integrins.
10 Chemical shifts (ppm) of 15 N/ 13 C-A20fmdv2 in PBS Residue N H N H α Others 1Asn H β2/β , 2.930; C β ; H δ21/δ , 7.766; N δ Ala C α ; H β 1.427; C β Val C ; C α ; H β 2.105; C β ; H γ1/γ , 1.055; C γ1/γ , Pro 5Asn C ; H β2/β , 2.871; C β ; H δ21/δ , 7.708; N δ Leu C ; C α ; H β2/β ; C β ; H γ 1.594; C γ ; H δ1/δ , 0.935; C δ1/δ , Arg C ; C α ; H β2/β , 1.931; C β ; H γ1/γ ; C γ ; H δ1/δ ; C δ ; H ε 7.471; N ε Gly C ; C α Asp C ; C α ; H β2/β ; C β Leu C ; C α ; H β2/β ; C β ; H γ 1.655; C γ ; H δ1/δ , 0996; C δ1/δ , Gln C ; C α ; H β2/β , 2.101; C β ; H γ1/γ ; C γ ; H ε21/ε , 7.651; N ε Val C ; C α ; H β 2.088; C β ; H γ1/γ , 1.014; C γ1/γ , Leu C ; C α ; H β2/β ; C β ; H γ 1.600; C γ ; H δ1/δ , 0.959; C δ1/δ , Ala C ; C α ; H β 1.407; C β Gln C ; C α ; H β2/β , 2.123; C β ; H γ1/γ ; C γ ; H ε21/ε , 7.631; N ε Lys C ; C α ; H β2/β , 2.365; C β ; H γ1/γ ; C γ ; H δ1/δ ; C δ ; H ε ; C ε Val C ; C α ; H β 2.084; C β ; H γ1/γ , 0.989; C γ1/γ , Ala C ; C α ; H β 1.412; C β Arg C ; C α ; H β2/β , 1.919; C β ; H γ1/γ ; C γ ; H δ1/δ ; C δ ; H ε 7.243; N ε Thr C ; C α ; H β 4.306; C β ; H γ ; C γ Hsl C ; C α ; H β2/β , 2.646; C β ; H γ1/γ , 4.588
11 Chemical shifts (ppm) of 15 N/ 13 C-DBD1 in PBS Residue N H N H α Others 1Glu 2Lys H β2/β ; H γ1/γ ; C γ ; H δ1/δ ; H ε ; C ε Cys 4Pro H β2/β , 2.325; H γ 2.068; H δ1/δ , 3.849; C δ Asn H β2/β , 2.880; C β ; H δ21/δ , 7.723; N δ Leu C α ; H β2/β ; C β ; H γ 1.631; C γ ; H δ1/δ , 0.969; C δ1/δ , Arg H β2/β , 1.950; C β ; H γ1/γ ; C γ ; H δ1/δ ; C δ ; H ε 7.471; N ε Gly C α Asp H β2/β , 2.765; C β Leu C α ; H β2/β ; C β ; H γ 1.638; C γ ; H δ1/δ , 0.978; C δ1/δ , Gln C α ; H β2/β , 2.146; C β ; H γ1/γ ; H ε21/ε , 7.682; N ε Val C α ; H β 2.150; C β ; H γ1/γ ; C γ1/γ Leu C α ; H β2/β ; C β ; H γ 1.636; C γ ; H δ1/δ , 0.982; C δ1/δ , Ala C α ; H β 1.447; C β Gln C α ; H β2/β , 2.179; C β ; H γ1/γ ; C γ ; H ε21/ε , 7.655; N ε Lys Val C α ; H β 2.127; C β ; H γ1/γ ; C γ Cys H β2/β , 3.282; C β Arg H β2/β , 1.932; C β ; H γ1/γ ; C γ H ε 7.307; N ε Thr C α ; H β 4.257; C β ; H γ ; C γ Hsl H β2/β , 2.267; H γ1/γ , 4.587
12 Chemical shifts (ppm) of 15 N 13 C-A20LAP2T1 Residue N H N H α Others 1Gly 2Phe 3Thr C α ; H β 4.255; C β ; H γ ; C γ Thr C α ; H β 4.299; C β ; H γ ; C γ Gly C α Arg C α ; H β2/β , 1.888; C β ; H γ 1.655; H δ1/δ ; H ε 7.321; N ε Arg C α ; H β2/β , 1.917; C β ; H γ1/γ ; H δ1/δ ; H ε 7.405; N ε Gly C α Asp H β2/β , 2.755; C β Leu C α ; H β2/β ; C β ; H γ 1.627; C γ ; H δ1/δ , 0.971; C δ1/δ , Ala C α ; H β 1.450; C β Thr C α ; H β 4.221; C β ; H γ ; C γ Ile C α ; H β 1.862; C β ; H γ12/γ , 1.417; C γ ; C δ His H β2/β , 3.266; C β Gly C α Leu C α ; H β2/β ; C β ; H γ 1.625; C γ ; H δ1/δ , 0.971; C δ1/δ , Asn H β2/β , 2.852; C β ; H δ21/δ , 7.711; N δ Arg H β2/β , 1.850; C β ; H γ1/γ ; H δ1/δ ; C δ ; H ε 7.258; N ε Pro C α ; H β2/β , 2.267; C β ; H γ1/γ ; C γ ; H δ1/δ , 3.785; C δ Phe H β2/β ; C β Hsl H β2/β , 2.529; H γ1/γ
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