Presentation on theme: "Supplementary Figure S1 (+)ABA NO (+)ABA Excess refractive index (x10 8 ) Molecular mass (kDa) 50±1 33±1 Excess refractive index (x10 8 ) Molecular mass."— Presentation transcript:
Supplementary Figure S1 (+)ABA NO (+)ABA Excess refractive index (x10 8 ) Molecular mass (kDa) 50±1 33±1 Excess refractive index (x10 8 ) Molecular mass (kDa) 37±2 49±1 59±2 33±3 PYL1 ΔNHAB1 Molecular mass (kDa) PYL1PYL1-HAB1 23±1 Excess refractive index (x10 8 ) 22±2 22±1 43±4 52±1 62±2 43±3 PYL6 ΔNHAB1 40±2 32±1 59±2 38±1 PYL8 ΔNHAB1 PYL6PYL6-HAB1 PYL8PYL8-HAB1 Dupeux et al., 2011 PYL6: 24kDa PYL1: 25.5kDa Elution volume (ml) PYL1 ΔNHAB1 PYL1 ΔNHAB1 PYL8 ΔNHAB1 PYL8 ΔNHAB1 PYL6 ΔNHAB1 PYL6 ΔNHAB1 ABA receptors exist in dimeric and monomeric forms. SEC-MALLS analysis of PYL1, PYL6 and PYL8 alone (left panels) and in the presence of ΔNHAB1 (right panels). The experiments were done in the absence (blue) and presence (red) of 1mM (PYL6 & PYL8) or 5mM ( PYL1) (+)ABA. The apparent size of PYL6 and PYL8 indicates that they are monomeric both in the presence and absence of (+)ABA. PYL1 is dimeric in the absence of ABA and addition of 5mM ABA produces partial dissociation. All receptor proteins tested in this study form 1:1 complexes when combined with ΔNHAB1 in the presence of (+)ABA (right panels). However, while dimeric proteins including PYL1 and PYR1 (see figure 2 in main text) do not interact with ΔNHAB1 in the absence of (+)ABA the formation of less stable complexes between monomeric receptors PYL6, PYL8 and ΔNHAB1 in the same conditions is revealed by a decrease in the height of the peaks corresponding to monomeric ΔNHAB1 and the appearance of new peaks containing both receptor proteins and ΔNHAB1 (figure 2 in main text shows similar experiments for PYR1 and PYL5). PYL8: 21.5kDa ΔNHAB1: 37kDa
Supplementary Figure S2Dupeux et al., 2011 A B 1 - 15 N (ppm) 2 - 1 H (ppm) p bound [ABA] / [PYR1] Determination of the dissociation constant, Kd, of the PYR1:ABA complex by solution NMR. (a) Part of 1 H- 15 N HSQC spectra of PYR1 with increasing amounts of ABA. The spectra correspond to the following [ABA]/[PYR1] ratios: 0.00 (red), 0.23 (blue), 0.47 (magenta), 0.70 (green), 2.59 (black). As the concentration of ABA increases, two resonances of D53, T124 and T125 are visible corresponding to free and ABA-bound PYR1, respectively. The assignment of the resonances was taken from Melcher et al. 2010. (b) Determination of the Kd value from the intensities of the double resonances of T125 (Kd=84 7 M) as described in Materials and Methods. Filled circles indicate experimental points, while the solid line corresponds to the least squares fit of Eq. 1. A weighted average over all three residues yields Kd=97 36 M.
Pro60 His60 (wt) Phe61 Lys51 Supplementary Figure S3Dupeux et al., 2011 Omit electron density map around proline 60 of the PYR H60P -ABA-HAB1 complex structure. The electron density confirms the substitution of histidine by proline at position 60. The structure of wt PYR has been superimposed on that of PYR1 H60P to help in the interpretation)
Supplementary Figure S4Dupeux et al., 2011 B. Sequence variation in regions determining the oligomeric structure of PYR/PYL proteins. Multiple sequence and secondary structure alignment of selected regions containing amino acids involved in ABA binding (red squares) and dimer formation (for PYR1,PYL1 and PYL2, green squares). PYR1, PYL1 and PYL2 are dimeric proteins while PYL5,PYL6 and PYL8 are monomeric. The structural and biochemical analysis of the PYR H60P mutant indicates that other proteins containing proline at the equivalent position, like PYL7, PYL9 and PYL10 are likely to be monomeric and show high intrinsic affinity for ABA. A.Structural proximity of amino acids involved in ABA binding and dimerization. Multiple sequence and secondary structure alignment of selected regions of dimeric PYR/PYL proteins. Residues involved in ABA binding (red squares) and dimer formation (green squares) are indicated. PYR1 PYL1 PYL2 60708090150
Your consent to our cookies if you continue to use this website.