supporting information - pnas...feb 26, 2009 · supporting information yi et al....
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Supporting InformationYi et al. 10.1073/pnas.0812432106
1 2 3 4 5 6
Fig. S1. Disruption of GDP-fucose de novo pathway and complementation with salvage pathway. Lane 1. LPS from wild-type E. coli O86; lane 2. Disruptionof de novo pathway (gmd-fcl); lane 3. complementation of de novo pathway with pTRC99A-f; lane 4. complemented with salvage pathway (pET15b-fkp), grownin LB without sugar supplement; lane 5. complemented with salvage pathway (pET15b-fkp), grown in LB supplemented with 0.1% glucose; lane 6. complementedwith salvage pathway (pET15b-fkp), grown in LB supplemented with 0.1% fucose.
Yi et al. www.pnas.org/cgi/content/short/0812432106 1 of 24
Fkp
Fig. S2. Expression and purification of Fkp. Lane 1. Protein Standard; lane 2. Proteins in whole cell of Fkp-expressing BL21(DE3); lane 3. Soluble protein fractions;lane 4. Inclusion body; lane 5. Flow-through from Ni-affinity purification; lane 6. Eluted Fkp from Ni-affinity purification.
Yi et al. www.pnas.org/cgi/content/short/0812432106 2 of 24
Fig. S3. In vitro Fkp reaction with fucose as substrate. Lane 1. ATP; lane 2. GTP; lane 3. L-fucose; lane 4. GDP-fucose; lane 5. ADP; lane 6. starting point ofGDP-fucose synthesis reaction; lane 7. GDP-fucose synthesis reaction after 30 min.
Yi et al. www.pnas.org/cgi/content/short/0812432106 3 of 24
A. Fucose, compound 1, product MS (ESI): 588.2
[1]Minutes
6 7 8 9 10 11 12 13 14 15 16
AU
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
AU
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
15
.41
75
31
1
84
.58
32
91
39
UV - 254nm0.1mM FourMix-254nm-pH10buf-01-5/5/2008 9:08:49 PM
Area PercentArea
GDP-FucATP
ADP
GTP
Minutes
6 7 8 9 10 11 12 13 14 15 16
AU
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
AU
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
15
.41
75
31
1
84
.58
32
91
39
UV - 254nm0.1mM FourMix-254nm-pH10buf-01-5/5/2008 9:08:49 PM
Area PercentArea
GDP-FucATP
ADP
GTP
B. compound 2, product MS (ESI): 574.1
Minutes
6 7 8 9 10 11 12 13 14 15 16
AU
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
AU
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
24
.25
96
65
9
75
.74
12
07
91
UV - 254nmD-Ara(2mM)_2hr_1.2ugE_9/8/2008 4:20:05 PM
Area PercentArea
Fig. S4. Capillary electrophoresis analysis of Fkp reactions with various fucose analogs.
Yi et al. www.pnas.org/cgi/content/short/0812432106 4 of 24
C. compound 3, product MS (ESI): 604.2
Minutes
6 7 8 9 10 11 12 13 14 15 16
AU
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
AU
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
17
.48
54
68
4
82
.51
52
21
05
UV - 254nmL-Gal(2mM)_2hr_1.2ugE_9/8/2008 3:20:05 PM
Area PercentArea
D. compound 4, product MS (ESI): 629.1
AU
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
AU
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
13
.64
93
45
9
86
.35
12
18
83
UV - 254nmFuc-N3(2mM)_2hr_1.2ugE_9/8/2008 3:53:07 PM
Area PercentArea
Fig. S4. (continued).
Yi et al. www.pnas.org/cgi/content/short/0812432106 5 of 24
E. compound 5, product MS (ESI): 603.2
6 7 8 9 10 11 12 13 14 15 16 17
AU
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
AU
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008UV - 254nmGal-NH2(2mM)_2hr_1.2ugE_9/8/2008 7:25:24 PM
Area PercentArea
F. compound 6, product MS (ESI): 602.1
Minutes
6 7 8 9 10 11 12 13 14 15 16 17 18
AU
-0.0010
-0.0005
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
0.0030
0.0035
0.0040
AU
-0.0010
-0.0005
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
0.0030
0.0035
0.0040
31
.66
46
73
4 68
.33
61
45
33
UV - 254nmR=CH2CH3_2hr_3.72ugE_9/15/2008 11:40:27 AM
Area PercentArea
Fig. S4. (continued).
Yi et al. www.pnas.org/cgi/content/short/0812432106 6 of 24
G. compound 7, product MS (ESI): 618.2
Minutes
6 7 8 9 10 11 12 13 14 15 16 17 18
AU
-0.0010
-0.0005
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
0.0030
0.0035
0.0040
AU
-0.0010
-0.0005
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
0.0030
0.0035
0.0040UV - 254nmR=CH(OH)CH3_2hr_3.72ugE_9/15/2008 11:09:11 AM
Area PercentArea
H. compound 8, product MS (ESI): 616.1
Minutes
6 7 8 9 10 11 12 13 14 15 16 17
AU
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
AU
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
9.2
23
27
16
90
.77
72
67
32
UV - 254nmKeto(2mM)_2hr_1.2ugE_9/8/2008 5:27:32 PM
Area PercentArea
Fig. S4. (continued).
Yi et al. www.pnas.org/cgi/content/short/0812432106 7 of 24
I. compound 9, product MS (ESI): 602.3
Minutes
6 7 8 9 10 11 12 13 14 15 16
AU
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
AU
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
10
.66
72
60
1
89
.33
32
17
83
UV - 254nmAldehyde(2mM)_2hr_1.2ugE_9/8/2008 6:27:44 PM
Area PercentArea
J. compound 10, product MS (ESI): 598.2
Minutes
6 7 8 9 10 11 12 13 14 15 16 17 18
AU
-0.0010
-0.0005
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
0.0030
0.0035
0.0040
AU
-0.0010
-0.0005
0.0000
0.0005
0.0010
0.0015
0.0020
0.0025
0.0030
0.0035
0.0040
17
.02
14
27
3
82
.97
92
08
32
UV - 254nmAlkyne_2hr_3.72ugE_9/15/2008 10:34:21 AM
Area PercentArea
Fig. S4. (continued).
Yi et al. www.pnas.org/cgi/content/short/0812432106 8 of 24
A. MS spectrum of LPS (compound 1). +
Q1:
3.5
76 to
3.9
43 m
in fr
om S
ampl
e 1
(Fuc
1; Q
1+ D
P 40
0) o
f IJL
XL08
0422
B03.
wiff
(Tur
bo S
pray
), su
btra
cted
(1.3
70 to
2.6
07 m
in),
...M
ax. 1
.1e6
cps
.
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
m/z
, am
u
1370
.110
81.0
1000
.991
5.8
1224
.063
0.6
934.
95%10%
15%
20%
25%
30%
35%
40%
45%
50%
55%
60%
65%
70%
75%
80%
85%
90%
95%
100%
Rel. Int. (%)
204.
3
468.
6
126.
1
528.
5
144.
144
8.4
186.
2
407.
536
6.5
138.
253
0.5
286.
332
7.3
651.
6
877.
930
4.4
388.
573
1.7
674.
685
5.9
152.
216
8.2
594.
637
0.6
489.
451
2.5
899.
829
1.4
243.
124
8.3
675.
634
8.2
550.
575
3.6
797.
761
0.6
503.
439
6.4
378.
335
7.3
273.
110
8.9
232.
3
Fig. S5. MS spectra of intact LPSs.
Yi et al. www.pnas.org/cgi/content/short/0812432106 9 of 24
B. MS spectrum of LPS (3)
+Q
1: 3
.576
to 3
.843
min
from
Sam
ple
3 (L
-Gal
1; Q
1+ D
P 4
00) o
f IJL
XL08
0422
B01
.wiff
(Tur
bo S
pray
), su
btra
cted
(2.3
06 to
3.0
08 m
in),.
..M
ax. 3
.5e5
cps
.
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
m/z
, am
u
5%10%
15%
20%
25%
30%
35%
40%
45%
50%
55%
60%
65%
70%
75%
80%
85%
90%
95%
100%
Rel. Int. (%)
136.
1
468.
615
2.1
144.
0
204.
3
878.
044
8.4
352.
4
530.
532
2.5
388.
5
509.
487
4.8
712.
641
0.4
248.
218
6.2
487.
411
9.1
856.
028
6.4
345.
352
8.5
295.
389
3.9
610.
565
1.6
915.
950
3.4
273.
373
0.6
223.
115
3.2
426.
359
5.7
217.
275
3.6
622.
549
3.4
975.
928
3.2
798.
017
7.0
443.
471
5.6
900.
138
4.2
547.
563
5.4
1038
.811
18.9
769.
512
80.9
1239
.8
Fig. S5. (continued).
Yi et al. www.pnas.org/cgi/content/short/0812432106 10 of 24
C. MS spectrum of LPS (2)
+Q
1: 3
.676
to 3
.910
min
from
Sam
ple
1 (D
Ara
-1; Q
1+ D
P 4
00) o
f IJL
XL0
8042
2B07
.wiff
(Tur
bo S
pray
), su
btra
cted
(1.5
04 to
2.3
73 m
in),.
..M
ax. 4
.2e5
cps
.
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
m/z
, am
u
5%10%
15%
20%
25%
30%
35%
40%
45%
50%
55%
60%
65%
70%
75%
80%
85%
90%
95%
100%
Rel. Int. (%)
126.
1
468.
5
204.
3
530.
5
144.
235
2.5
388.
585
6.0
878.
013
6.1
286.
232
7.3
448.
418
6.3
322.
515
2.1
304.
448
7.2
248.
340
7.4
370.
665
1.6
122.
171
2.6
509.
434
5.3
295.
241
0.5
550.
648
9.4
109.
026
6.2
503.
366
0.6
429.
526
1.2
892.
958
0.5
838.
131
2.4
730.
775
3.6
975.
918
1.2
1279
.423
9.2
552.
646
6.5
642.
412
40.9
1098
.044
0.6
1077
.911
5.1
842.
158
3.5
1339
.010
59.5
923.
793
3.7
Fig. S5. (continued).
Yi et al. www.pnas.org/cgi/content/short/0812432106 11 of 24
D. MS spectrum of LPS (4)
+Q
1: 3
.442
to 3
.643
min
from
Sam
ple
1 (F
uc-N
3 1;
Q1+
DP
400
) of I
JLX
L080
422B
06.w
iff (T
urbo
Spr
ay),
Sm
ooth
edM
ax. 1
.1e5
cps
.
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
m/z
, am
u
5%10%
15%
20%
25%
30%
35%
40%
45%
50%
55%
60%
65%
70%
75%
80%
85%
90%
95%
100%
Rel. Int. (%)
126.
1
468.
6
204.
2
322.
4 352.
5
144.
1
138.
0
304.
3
327.
387
4.8
186.
338
8.5
878.
024
3.2
334.
415
2.1
712.
644
8.4
370.
513
3.0
201.
228
6.5
651.
685
5.9
509.
534
5.2
550.
541
0.4
489.
622
5.2
730.
867
1.6
291.
215
7.3
837.
952
9.4
313.
281
9.8
611.
489
3.0
402.
249
5.5
701.
447
9.4
660.
497
6.7
1078
.164
2.7
763.
795
1.0
865.
012
81.1
557.
412
17.0
989.
211
46.9
1441
.913
32.9
Fig. S5. (continued).
Yi et al. www.pnas.org/cgi/content/short/0812432106 12 of 24
E. MS spectrum of LPS (5)
+Q1:
3.509
to 3.
810 m
in fro
m Sa
mple
1 (U.
Ohio
, CPS
-GalN
, DP=
400V
) of IJ
LJL0
8042
9A01
.wiff
(Turb
o Spra
y), S
mooth
edMa
x. 1.3
e6 cp
s.
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
m/z,
amu
878.0
1.00e
5
2.00e
5
3.00e
5
4.00e
5
5.00e
5
6.00e
5
7.00e
5
8.00e
5
9.00e
5
1.00e
6
1.10e
6
1.20e
6
1.30e
6
Intensity, cps
136.2 15
2.2
348.3
428.3
330.3
312.3
468.6
232.3
122.1
524.4
250.2
622.4
410.2
322.5
204.3
144.1
458.4
538.5
856.0
241.2
659.6
604.2
401.5
750.5
838.0
377.2
Fig. S5. (continued).
Yi et al. www.pnas.org/cgi/content/short/0812432106 13 of 24
F. MS spectrum of LPS (6)
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z, amu
152.1
136.0
468.5
126.0428.3330.2
348.3312.3204.2
855.9388.5410.2144.1874.6248.3273.2 530.4487.3 622.4370.4119.0153.2
712.6284.3239.2 503.3 892.7297.2 616.5440.4 837.8730.4659.5385.2181.3 975.9741.4 1077.7 1338.91146.9 1281.8
Fig. S5. (continued).
Yi et al. www.pnas.org/cgi/content/short/0812432106 14 of 24
G. MS spectrum of LPS (7)
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z, amu
468.6
152.2
126.1
322.4
204.0 874.6352.3144.2 388.5
856.0330.2312.2 428.3487.3530.4185.9 712.6304.2168.2 250.4
355.3 508.5550.5692.6448.1 820.0232.0 286.2 892.9 976.0
Fig. S5. (continued).
Yi et al. www.pnas.org/cgi/content/short/0812432106 15 of 24
H. MS spectrum of LPS (8)
+Q
1: 4
.311
to 4
.846
min
from
Sam
ple
1 (W
ang
Keto
; Q1+
DP
400)
of I
JLXL
0808
25A0
8.w
iff (T
urbo
Spr
ay),
subt
ract
ed (2
.172
to 2
.573
...M
ax. 9
.8e5
cps
.
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
m/z
, am
u
5.0e
4
1.0e
5
1.5e
5
2.0e
5
2.5e
5
3.0e
5
3.5e
5
4.0e
5
4.5e
5
5.0e
5
5.5e
5
6.0e
5
6.5e
5
7.0e
5
7.5e
5
8.0e
5
8.5e
5
9.0e
5
9.5e
59.
8e5
Intensity, cps
468.
5
316.
5
855.
8
204.
3
322.
3
366.
387
7.8
388.
442
8.3
352.
4
330.
234
8.1
312.
252
4.3
569.
440
7.3
288.
448
7.2
622.
230
4.3
448.
569
2.5
712.
533
4.4
248.
224
3.2
837.
851
5.3
426.
428
4.1
591.
497
5.8
671.
648
0.5
893.
855
0.4
819.
762
9.4
411.
736
2.1
229.
210
77.8
915.
626
8.2
997.
686
9.4
730.
588
6.5
682.
254
0.3
Fig. S5. (continued).
Yi et al. www.pnas.org/cgi/content/short/0812432106 16 of 24
I. MS spectrum of LPS (9)
+Q
1: 4
.077
to 4
.512
min
from
Sam
ple
1 (W
ang
Alde
hyde
; Q1+
DP
400)
of I
JLXL
0808
25A0
9.w
iff (T
urbo
Spr
ay),
subt
ract
ed (1
.504
to 2
...M
ax. 3
.2e5
cps
.
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
m/z
, am
u
2.0e
4
4.0e
4
6.0e
4
8.0e
4
1.0e
5
1.2e
5
1.4e
5
1.6e
5
1.8e
5
2.0e
5
2.2e
5
2.4e
5
2.6e
5
2.8e
5
3.0e
5
3.2e
5
Intensity, cps
330.
231
6.7
428.
3
468.
531
2.2
348.
025
0.0
622.
252
4.3
874.
641
0.3
344.
028
8.4
204.
3
850.
4
509.
238
8.3
419.
033
4.0
855.
840
7.3
604.
152
7.3
356.
929
8.6
490.
654
0.3
217.
267
1.6
457.
075
7.4
515.
083
7.6
450.
425
7.2
324.
559
5.3
692.
238
3.1
659.
357
2.5
265.
544
2.9
773.
323
5.2
472.
391
5.7
537.
469
7.1
730.
383
3.4
562.
465
6.2
395.
1
Fig. S5. (continued).
Yi et al. www.pnas.org/cgi/content/short/0812432106 17 of 24
J. MS spectrum of LPS (10)
+Q
1: 4
.077
to 4
.445
min
from
Sam
ple
1 (W
ang
Alky
ne; Q
1+ D
P 40
0) o
f IJL
XL08
0825
A10.
wiff
(Tur
bo S
pray
), su
btra
cted
(2.3
39 to
2.8
...M
ax. 5
.9e5
cps
.
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
m/z
, am
u
5.0e
4
1.0e
5
1.5e
5
2.0e
5
2.5e
5
3.0e
5
3.5e
5
4.0e
5
4.5e
5
5.0e
5
5.5e
5
5.9e
5
Intensity, cps
468.
5
877.
8
855.
934
4.3
878.
942
8.3
204.
241
0.5
352.
3
388.
448
7.2
366.
371
2.5
524.
449
0.5
288.
431
2.0
550.
540
7.3
392.
850
8.4
248.
442
9.4
753.
630
4.3
1077
.897
5.8
621.
859
1.5
334.
165
1.6
693.
550
7.2
859.
844
8.3
397.
391
5.7
299.
128
4.2
730.
637
5.1
222.
979
7.8
707.
156
8.2
629.
4
Fig. S5. (continued).
Yi et al. www.pnas.org/cgi/content/short/0812432106 18 of 24
10 9 1 8 7 6 5 4 3 2 1
OHO
OH
OHHO
OHO
OH
OHHO
OHO
OH
OHHO
OHO
OH
OHHO
OHO
OH
OHHO
HO N3 H2N
OHO
OH
OHHO
OHO
OH
OHHO
HO OHO
OH
OHHO
O OHO
OH
OHHO
O
H
OHO
OH
OHHO
54321
19876 0
Fig. S6. Analysis of LPSs fed with different fucose analogs. Chemical structures of compounds are shown.
Yi et al. www.pnas.org/cgi/content/short/0812432106 19 of 24
A. compound 5
30 35 40 45 50 55 60 650 70 75 80 850 90 95 100 105 110 115
m/z, amu
20
40
60
80
100
120
140
160
180
200
220
Intensity, cp
529.
570.
691.0366.
732.
407.330.5
652.
490. 853.0
312.608.5497.0 675.0 814.350. 401. 507.5416. 708. 878.0 1056.614. 801. 895.0858.5469.335.0 720.5358. 976.0541.
Fig. S7. Precursor-treated MS spectra.
Yi et al. www.pnas.org/cgi/content/short/0812432106 20 of 24
B. compound 6
300 350 400 450 500 550 600 650 700 750 800 850 900 9501000 0031 0521 0021 051100110501m/z, amu
732.0
689.0407.5
366.5
892.0
570.0
853.0651.5
935.0
489.0508.0330.0 801.5 1095.0608.0 814.0873.0
732.
689.691.
651.
Fig. S7. (continued).
Yi et al. www.pnas.org/cgi/content/short/0812432106 21 of 24
C. compound 7
30 35 40 45 50 55 60 65 70 75 80 85 900 95 100 105 110 115 120 125 130m/z, amu
529.
691.
853.366.
732.570.407. 651.5675.609.490. 878.
801.307. 446. 672. 870.547. 638. 894.388. 709.513.
Fig. S7. (continued).
Yi et al. www.pnas.org/cgi/content/short/0812432106 22 of 24
D. compound 10
+Prec (204.20): 4.380 to 4.948 min from Sample 1 (Wang Alkyne; DP 400, pre@204) of IJLXL080825A11.wiff (Turbo Spray), Max. 3474.0
30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130m/z,
20
40
60
80
100
120
140
160
180
200
220
240
260
280
300
320
340
Intensity, cp
529.
685.
732.
888.
570.
330. 652.
490. 853.312.814.408. 670.
367. 428.348. 609. 920.472.389. 716.524.460.401. 873. 976.776. 1017.701.320. 794.641.
Fig. S7. (continued).
Yi et al. www.pnas.org/cgi/content/short/0812432106 23 of 24
Other Supporting Information Files
SI Appendix
fucN3 + + + −fuc − − − +Cu2+ + − + +TCEP + + − +Ligand + + + +
1 2 3 4
Fig. S8. Control experiments for in vitro polysaccharide labeling.
Yi et al. www.pnas.org/cgi/content/short/0812432106 24 of 24