Transcript Slide 1

Nutrient Transport in E. coli
Ferrichrome
Vitamin B12
FhuA
BtuB
FhuD
BtuF
FhuB/C
BtuC/D
Passive transporters : Most nutrients < 600 Da - Porins
Ligand Gated Porins - sugars
Active transporters : Larger nutrients such as iron and cobalamin (vitamin
B12 : CNCbl) BtuB, FecA, FhuA, and FepA etc.
Proposed TonB Dependent Transport Cycle
Extracellular Space
CNCbl
A
B
CNCbl
C
D
A
BtuB
Outer
Membrane
CNCbl
Periplasmic
Space
Ton box
TonB
CNCbl
ExbB
Inner
Membrane
ExbD
Cytoplasmic Space
DmH+
EPR Spectra for 2 consecutive strands of the barrel
L2 187
L3
T G
P S
A
L
R L9
Q NT
L10
L
G
A G
490 A A
534
D
P
403
I
L7
L4
Q
285
Y S
D 444 L8
L6
T
S
G YL
Y
T AY
361
P
S DT
V D I
D
R
Y
576 L11
F
R
D
Y
Y
Y Y
L
Q N D
S
325 L5
V T
G L K AY N VV
Q
D PT
D
D GY G
S
Y
S SD T G
Y DD S
L
A HY Y
D GD D
G
E V T DK L
N
Q
S
V
N F
R
G
P
T
S
Y F
Y
Y
Y M
P
Y RL
V T H D NP
P
A
Y
T
L
Q
N
G
L1
Q
D
A RG A
V
T
D YD S
T SL
H S N
T
K GG T R R HT
L RN L
K H
G
E
T
E
W
R K
T
A
D V
A
K
D
N
Y
G
T
Y P
Y G
E
D
Q
M
D
N
H
S
G
F
Y
S
L QK L
S
F
G
K
K DY A
D L T A NQ G
R
L
Y SG E
T
E
N WL Q Y Q G P
N
K G T W F T D Y WQ Y Q K L G G Y Y D Y Y G W
I
Y
A D
S
G G
D
Y KV T GE R K S
Y TQ D
Q
D
T
R
Y
T
I
V SA
G
R
V
V SH Y
G LA L
L
K AL I T
Y F D K NW Q A S
S
S
V
N
G
Q
N
R
L
I
S
G V
Q N
A
W I
A
Y G
T
G
V HE
T
E
G
R
L
R GV E
G QQ GT GA AW S
F
E
F
V QQ T
S
V YA D W D T A
I
G
W
T
N
A A
R
L
G F
R
Y
T
W
K
E GL Y I S I NN I
Q PG
Y
I
S
T
T
L VP DF
Q AA G L T
F
L
G I F
L
G
V
K
W DT
K GL
E
F
H
L
V
E
T
Y
E
G
E
H
I
Q S
A
D
D 135
S T
594
343
264
V
465
424
162
512
381
214
G
R
557
304
8
7
6
5
4
3 2 1 TT
22 21 20 19 18
17 16 15 14 13 12 11 10 9
I I
N V V G G
I
A
D
S
G
G
V
R
L
N
L
A
G
V
S
G
S
A
D
L
S
Q
F
P
I
A
L
V
Q
R
V
E
Y
I
R
G
P
R
S
A
V
Y
D
I L V L V H S A N T G R I S S L Q G S G G N Q T I D V G P L R R L V D N V S
T
Q D T S P D T L V V T A N R F E Q P R S T V L A P T T V V T R Q D I D R W Q S
H176R1
S148R1
T175R1
Y149R1
H174R1
Q150R1
A173R1
N151R1
Y172R1
Y152R1
D171R1
D153R1
G170R1
V154R1
240
Fanucci et al. Biochemistry, 41, 11543, 2002
S155R1
L168R1
T156R1
T167R1
Q157R1
V166R1
Q158R1
R165R1
Q159R1
T164R1
L160R1
x1.5
K163R1
G161R1
x1.5
D162R1
x2
Accessibility is measured via Relaxation Measurements
Strategy: Add a paramagnetic species that can collide with spin label
~ relative enhancements in relaxation times
Enhancements in relaxation due to paramagnet tell you
about collisional frequency and hence accessibility
•Pulsed EPR - direct T1 measurements
Vs
•Continuous Wave Power Saturation - indirect T1 measurements
Nitroxide Accessibility and Membrane Depth from EPR
Power Saturation of the EPR Resonance
accessibility
Poxy = P´1/2oxy - P´1/2nit
Pmet = P´1/2met - P´1/2nit
metal
A
membrane
oxygen
 = ln (DP1/2oxy/DP1/2metal)
A = I•P1/2 •[1 + (21/e - 1)P/P1/2]-e
P1/2 = 19.1
met
4
2
oxy
P1/2 = 10.6
Aqueous

0
nit
Hydrocarbon
P1/2 = 5.6
-2
0
2
4
6
Sqrt (P)
8
10
-40
-20
0
20
Distance from phosphate (A)
40
Calibration Points:
Sample
bR and DOXYL-PC
 Distance
5-DOXYL
7-DOXYL
10-DOXYL
12-DOXYL
+1.5
+2.1
+2.4
+2.8
8.1
10.5
14.0
16.0
bR109R1
bR116R1
bR117R1
bR124R1
+2.2
+4.1
+4.2
+2.0
11.0
20.5
22.5
10.5
PDB ID: 1ap9
Frazier, A.A. et al. Biochem, 42 (2003), 96-105
Modeling of C2A on the Membrane Surface
4
C2 domain
doxyl-PC
bacteriorhodopsin

2
0
-2
-40
229
142 171
24
239
202
197b
0 170
201
176
231
237
233
173
236
175
199197
234
174
-20
0
20
40
Distance from Phosphate (A)
  A tanh B distance - C  D
Frazier, A.A. et al. Biochem, 42 (2003), 96-105
BtuB as a Ruler From the Bilayer Surface
DOGS-NTA-Ni
Ni(II)EDDA
Ni(II) ca. 14 Å
BtuB as a Ruler From the Bilayer Surface
membrane
O2
O2
Ni(II) ca. 14 Å
aqueous
DOGS-Ni-NTA
O2
Ni(II)EDDA
12
4
8
2
4
0
0

6
aqueous
-2
membrane
-20
-10
0
10
20
30
Distance from Phosphates (Å)
 = ln (DP1/2oxy/DP1/2metal)
DP1/2 DOGS-Ni-NTA
Ni(II)EDDA
Conclusions
Ni(II)EDDA
SDSL “Rulers”
C2A CPLA2
DOGS-Ni-NTA
~ 14Å
O2
bR
BtuB
KcsA
POPC + BMP
GM2AP
~ 2Å
A60
GM2AP oligomerization
Lipid Composition Affects Binding
Secondary Structure Analysis: Spin Label Mapping
Transmembrane Water Filled Pores
a-helix bundle
accessibility
P
oxygen
metal complex
membrane
aqu
b-barrel
2
membrane
aqueous phase
4
6
P
8
oxygen
metal complex
membrane
2
4
6
8
Power Saturation of EPR Spectra for 2
consecutive putative strands of the b-barrel
2.0
0.2
1.6
0.1
1.2
0.0
aqu
0.8
membrane
-0.2
0.0
146 148 150 152 154 156 158 160 162 164
2.0
0.2
1.6
0.1
1.2
0.0
0.8
-0.1
0.4
-0.2
0.0
178 176 174 172 170 168 166 164 162
residue
26 of 36
Oxygen accessibility
Ni(acac)2 accessibility
-0.1
0.4
b-barrel
aqueous phase
membrane
Power Saturation of EPR Spectra for 2
consecutive putative strands of the b-barrel
2.0
0.2
1.6
0.1
1.2
Residue
0.0
148
3
0.8
150
152
154
156
158
160
3
146 148 150 152 154 156 158 160 162 164
2.0
0.2
1.6
0.1
2
2
1
1
0
0
membrane
aqueous
-1
1.2
0.0
0.8
-0.1
0.4
-0.2
0.0

0.0

-0.2
Oxygen accessibility
Ni(acac)2 accessibility
-0.1
0.4
-1
-2
-2
172
170
168
166
164
162
Residue
178 176 174 172 170 168 166 164 162
residue
27 of 36
Fanucci et al. Biochemistry, 41, 11543, 2002
Structure of BtuB
Side View
Bottom View
S1 (25-31)
Extracellular
Tonbox
(6-12)
Lipid Bilayer
Periplasmic
extended
tonbox (13-17)
• 66 kD protein
• beta barrel of 22 antiparallel strands
• globular hatch/core domain
Chimento et al. NSB 2003 (10) 394-401