A Theoretical study on Negative Refractive Index Metamaterials (Review)
Supplementary Figure S1 (+)ABA NO (+)ABA Excess refractive index (x10 8 ) Molecular mass (kDa) 50±1...
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Transcript of Supplementary Figure S1 (+)ABA NO (+)ABA Excess refractive index (x10 8 ) Molecular mass (kDa) 50±1...
Supplementary Figure S1
(+)ABA
NO (+)ABA
Exc
ess
ref
ract
ive
ind
ex
(x1
08)
Exc
ess
ref
ract
ive
ind
ex
(x1
08)
Mo
lecu
lar
mas
s (k
Da
)
14 16 18 20
0
300
600
900
1200
1500
1800
50±133±1
Exc
ess
ref
ract
ive
ind
ex
(x1
08 )
14 16 18 20
1
10
100
Mo
lecu
lar
mas
s (k
Da
)
37±249±159±2
33±3
PYL1
ΔNHAB1
Mo
lecu
lar
mas
s (k
Da
)
PYL1 PYL1-HAB1
14 16 18 20
0
100
200
300
400
500
600
23±123±1
Exc
ess
ref
ract
ive
ind
ex
(x1
08)
14 16 18 20
0
200
400
600
800
1000
22±222±1
14 16 18 201
10
100
43±452±162±243±3
PYL6
ΔNHAB1
14 16 18 20
1
10
100
40±232±159±238±1
PYL8
ΔNHAB1
PYL6 PYL6-HAB1
PYL8 PYL8-HAB1
Dupeux et al., 2011
PYL6: 24kDa
PYL1: 25.5kDa
Elution volume (ml) 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 S2 Dupeux et al., 2011
10.2 9.8 9.4
125
120
115
110
1
- 15
N (
pp
m)
10.2 9.8 9.4 10.2 9.8 9.4
2 - 1 H (ppm)
10.2 9.8 9.4 10.2 9.8 9.4
125
120
115
110D 5 3
T 1 2 5
T 1 2 4
a
b
[A B A ]/[P Y R 1 ]
p boun
d
0 .0 0 .5 1 .0 1 .5 2 .0 2 .5 3 .00 .0
0 .2
0 .4
0 .6
0 .8
1 .0T 1 2 5
A
B
1-
15N
(pp
m)
2- 1H (ppm)
p boun
d
[ABA] / [PYR1]
Determination of the dissociation constant, Kd, of the PYR1:ABA complex by solution NMR. (a) Part of 1H-15N 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=847M) 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=9736M.
Pro60
His60(wt)
Phe61
Lys51
Supplementary Figure S3 Dupeux et al., 2011
Omit electron density map around proline 60 of the PYRH60P-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 PYR1H60P to help in the interpretation)
Supplementary Figure S4 Dupeux 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 PYRH60P 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
PYR1PYL1
PYL2
60 70 80 90 150